Human Autotaxin ELISA Kit (96 Tests). Quantitate Human ENPP2 in cell culture supernatants, serum, plasma (heparin) , urine and human milk. Sensitivity: 10pg/ml.
Product Type:
Assay & Detection
Storage Temp:
Mixed
Immunogen:
Expression system for standard: NS0; Immunogen sequence: A36-I863
Applications:
ELISA
Additional Info:
For Quantitative Detection of human Autotaxin in cell culture supernatants, serum, plasma (heparin), urine and human milk.
Biosite Brand:
BioSite ELISA
Species Reactivity:
human
Cross Reactivity:
There is no detectable cross-reactivity with other relevant proteins.
Human CA3 ELISA Kit (96 Tests). Quantitate Human CA3 in cell culture supernatants, serum and plasma (heparin, EDTA, citrate) and urine. Sensitivity: 50pg/ml.
Product Type:
Assay & Detection
Storage Temp:
Mixed
Immunogen:
Expression system for standard: E.coli; Immunogen sequence: A2-K260
Applications:
ELISA
Additional Info:
For quantitative detection of human CA3 in cell culture supernatants, serum and plasma (heparin, EDTA, citrate) and urine.
Biosite Brand:
BioSite ELISA
Species Reactivity:
human
Cross Reactivity:
There is no detectable cross-reactivity with other relevant proteins.
Human CRLF2/TSLP R ELISA Kit (96 Tests). Quantitate Human CRLF2 in cell culture supernatants, cell lysates, serum and plasma (heparin, EDTA). Sensitivity: 10pg/ml.
Product Type:
Assay & Detection
Storage Temp:
Mixed
Immunogen:
Expression system for standard: NS0; Immunogen sequence: G25-K231
Applications:
ELISA
Additional Info:
For quantitative detection of human CRLF2 in cell culture supernatants, cell lysates, serum and plasma (heparin, EDTA).
Biosite Brand:
BioSite ELISA
Species Reactivity:
human
Cross Reactivity:
There is no detectable cross-reactivity with other relevant proteins.
Rat SEMA3F ELISA Kit (96 Tests). Quantitate Rat SEMA3F in cell culture supernatants, serum and plasma (heparin, EDTA or citrate). Sensitivity: 10pg/ml.
Product Type:
Assay & Detection
Storage Temp:
Mixed
Immunogen:
Expression system for standard: NS0; Immunogen sequence: A19-T785
Applications:
ELISA
Additional Info:
For quantitative detection of rat SEMA3F in cell culture supernatants, serum and plasma (heparin, EDTA, citrate).
Biosite Brand:
BioSite ELISA
Species Reactivity:
rat
Cross Reactivity:
There is no detectable cross-reactivity with other relevant proteins.
Human Ficolin-1 / FCN1 / M Ficolin ELISA Kit (96 Tests). Quantitate Human FCN1 in cell culture supernatants, serum and plasma (heparin). Sensitivity: 20pg/ml.
Product Type:
Assay & Detection
Storage Temp:
Mixed
Immunogen:
Expression system for standard: NS0; Immunogen sequence: A30-A326
Applications:
ELISA
Additional Info:
For quantitative detection of human Ficolin-1 in cell culture supernatants, serum and plasma(heparin).
Biosite Brand:
BioSite ELISA
Species Reactivity:
human
Cross Reactivity:
There is no detectable cross-reactivity with other relevant proteins.
Human TLR1 / TIL ELISA Kit (96 Tests). Quantitate Human TLR1 in cell culture supernatants, cell lysates, serum and plasma (heparin, EDTA). Sensitivity: 10pg/ml.
Product Type:
Assay & Detection
Storage Temp:
Mixed
Immunogen:
Expression system for standard: NS0; Immunogen sequence: S22-N578
Applications:
ELISA
Additional Info:
For quantitative detection of human TLR1 in cell culture supernatants, cell lysates, serum and plasma (heparin, EDTA).
Biosite Brand:
BioSite ELISA
Species Reactivity:
human
Cross Reactivity:
There is no detectable cross-reactivity with other relevant proteins.
Our purified lyophilized exosomes are obtained from different biological sources including cell culture supernatant, human plasma, serum and urine. Isolation is performed by a combination of ultracentrifugation and microfiltration procedures, and subsequent quantification/validation for overall protein content and particle number by NTA with Nanosight.
Background Info:
Exosomes are small endosome derived lipid nanoparticles (50-120 nm) actively secreted by exocytosis by most living cells. Exosome release occurs either constitutively or upon induction, under both normal and pathological conditions, in a dynamic, regulated and functionally relevant manner. Both amount and molecular composition of released exosomes depend on the state of a parent cell. Exosomes have been isolated from diverse cell lines (hematopoietic cells, tumor lines, primary cultures, virus infected cells) as well as from biological fluids in particular blood (e.g. serum and plasma from cancer patients) and other body fluids (bronchoalveolar lavage fluid, pleural effusions, synovial fluid, urine, amniotic fluid, semen, saliva etc). Exosomes have pleiotropic physiological and pathological functions and an emerging role in diverse pathological conditions such as cancer, infectious and neurodegenerative diseases.
Product Type:
Lyophilized exosomes
Storage Temp:
Store up to 3 years at 4°C >>> Storage of reconstituted exosomes: -20°C for up to one month or -80°C for up to 6 months. Avoid repeated freeze-and-thaw cycles.
Lyophilized Exosome Standards from MSC from human adipose tissue (for research use only)
Additional Info:
Lyophilization is an ideal method for long-term storage of exosomes and microvesicles. It does not alter the stability of exosome proteins and nucleic acids, in comparison to other storage methods, including storage of fresh EVs at -20°C. Lyophilized EVs and microvesicles are easy to ship and stable for long term storage (up to 36 months).
Application Details:
Assay calibration. Control (spike-in) for exosome quantification. Protein marker analysis using different techniques. Extraction and analysis of exosome nucleic acid. Standardized positive controls for immunocapture performance evaluation. Flow cytometry. Electron microscopy.
The antibody labels a 50kDa, multiple myeloma oncogen-1 (MUM1) protein. MUM1 is encoded by the MUM1/IRF-4 gene, which is mapped to 6q23-25 and identified as a myeloma-associated oncogene. It is a member of the interferon regulatory factor family of transcription factors and plays an important role in the regulation of gene expression in response to signaling by interferon and other cytokines. MUM1 positive cells express the protein in the nucleus in a diffuse and microgranular pattern. However, some positivity is also observed in the cytoplasm of MUM1-expressing cells. In normal/reactive lymphoid tissues, such as lymph node, this antibody stains plasma cells, some B-cells in the light zone of germinal centers, and a subset of T-cells (T-cells in germinal centers and interfollicular areas). MUM1 expression has been described in diffuse large B-cell lymphoma (DLBCL). MON 3318 can stain other Bcell lymphomas such as lymphoplasmacytic lymphoma, chronic lymphocytic leukemia, follicular lymphoma, marginal zone lymphoma, lymphoblastic lymphoma/leukemia, primary effusion lymphoma, DLBCL, Burkitt-like lymphoma, and classical Hodgkin lymphoma.
Antibody Isotype:
IgG1
Monosan Range:
MONOSAN
Clone:
MRQ-8
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Falini B, et al. Blood. 2000; 95:2084-92
References 2:
Grossman A, et al. Genomics. 1996; 37:229-33
References 3:
Neresh KN. Haematologica. 2007; 92:267-8
References 4:
Van Imhoff GW, et al. J Clin Oncol. 2006; 24:4135-42
References 5:
Gualco G, et al. Appl Immunohistochem Mol Morphol. 2010; 18:301-10
Common acute lymphoblastic leukemia antigen (CALLA / CD10) is a useful marker for the characterization of childhood leukemia and B cell lymphomas. This antibody reacts with antigen of lymphoblastic, Burkitts, and follicular lymphomas; and chronic myelocytic leukemia. Also, Anti-CD10 detects the antigen of glomerular epithelial cells and the brush border of the proximal tubules; this characteristic may be helpful in interpreting renal ontogenesis in conjunction with other markers. Other non-lymphoid cells that are reactive with CD10 are breast myoepithelial cells, bile canaliculi, neutrophils and small population of bone marrow cells, fetal small intestine epithelium, and normal fibroblasts.
Antibody Isotype:
IgG1
Monosan Range:
MONOSAN
Clone:
56C6
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Maguer-Satta V, et al.. Stem Cells. 2011; 29:389-96
Nerve growth factor receptor (NGFR), also known as p75NTR, is a 75-kDa glycoprotein member of the tumor necrosis factor (TNF) receptor family essential for embryonic development of the peripheral nervous system. In normal tissue, NGFR is expressed in neural crest derived cells, lymphoid follicular dendritic cells seen in lymph nodes and tonsils, and myoepithelial cells of the breast, prostate, and salivary gland as well as basal epithelium of the respiratory system. NGFR has been shown to be a reliable adjunct marker for melanoma, specifically desmoplastic and spindle cell variants Anti-NGFR labels myoepithelial cells of the breast and may aid in the differentiation between benign conditions, pre-invasive neoplastic lesions and invasive malignancies of the breast.
Antibody Isotype:
IgG1
Monosan Range:
MONOSAN
Clone:
MRQ-21
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Thompson SJ. Am J Clin Pathol. 1989; 92:415-23
References 2:
Reis-Filho JS, et al. Mod Pathol. 2006; 19:307-19
References 3:
Lazova R, et al. J Am Acad Dermatol. 2010; 63:852-8
SOX-11 which is a member of the SOX (SRY-related HMG-box) family is a transcription factor normally expressed in the developing human central nervous system and plays a role in embryonic cell determination. Studies show that SOX-11 can be used as a marker for mantle cell lymphoma (MCL). Mantle cell lymphoma (MCL) accounts for 5% to 10% of mature B-cell neoplasms and is an aggressive disease genetically characterized by overexpression of cyclin D1 (CCND1), an important regulator of the G1/S phase of the cell cycle, due to the specific translocation t(11;14)(q13;q32).
Antibody Isotype:
IgG1
Monosan Range:
MONOSAN
Clone:
MRQ-58
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
The Plus HRP Polymer anti-Mouse kit is designed for the qualitative detection of antigens in fixed paraffinembedded tissue sections, in frozen tissue sections, and in cytological samples. It was developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The reagent can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Polymer anti-Mouse kit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer consists of several molecules of secondary antibodies covalently bound to several molecules of horse radish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of monoclonal primary antibodies and sera obtained from mice. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP Polymer antiMouse kit avoids the problem of background staining caused by endogenous biotin in the tissue
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRPpolymer is minimized by incubation with a protein blocking solution. This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the HRP-polymer is applied and incubated. Any excess of unbound HRP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
100 mL HRP-Polymer anti-Mouse (ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB High contrast kit. Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagent should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. HRP-polymer anti-Mouse 30 min. 8. Washing with wash buffer 3 x 2 min. 9. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution can result in decreasing signal intensity. Sanbio guarantee that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of the reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining might appear. ProClin 950 is used for stabilisation. A Material safety data sheets (MSDS) is available upon request.
p63 consists of two major isoforms -TAp63 and ?Np63. These isoforms differ in the structure of the N-terminal domains. The TAp63 isoform (identified by anti-p63 antibody) contains a transactivation- competent TAdomain with homology to p53, which regulates the expression of the growth -inhibitor y genes. In contrast, ?Np63 isoform (identified by anti-p40 antibody) contains an alternative transcriptionally- inactive ?N domain, which antagonizes the activity of TAp63 and p53. The p40 (clone ZR8) recognizes exclusively ?Np63 but not TAp63. p40 is a squamous cell carcinoma specific antibody. It reacts with the vast majority of cases of squamous cell carcinomas of various origins, but not with adenocarcinomas. It is particularly useful in differentiating lung squamous cell carcinoma from lung adenocarcinoma. p40 antibody can also be used as an alternative basal cell/myoepithelial cell marker, which has similar sensitivity and specificity as that of p63 antibody. Therefore, p40 antibody may also be used as an alternative immunohistochemical marker for determining prostate adenocarcinoma vs. benign prostate glands and for determining breast intraductal carcinoma v s. invasive breast ductal carcinoma. Pretreatment: Heat induced epitope retrieval in 10 mM citrate buffer, pH6.0, or in 50 mM Tris buffer pH9.5, for 20 minutes is required for IHC staining on formalin-fixed, paraffin embedded tissue sections. Note: Dilution of the antibody in 10% normal goat serum followed by a goat anti-rabbit secondary antibody-based detection is recommended. Control tissue Lung squamous cell carcinoma. Staning nuclear
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
ZR8
Concentration:
n/a
Format:
Purified
Storage buffer:
Purified antibody in 0.2% BSA and 15mM sodium azide.
The Plus HRP One-Step Polymer anti-Mouse/Rabbit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It was developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP One-Step Polymer anti-Mouse/Rabbit is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer consists of several molecules of secondary antibodies covalently bound to several molecules of horseradish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP One-Step Polymer kits avoid the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRP One-Step Polymer is minimized by incubation with a protein blocking solution. This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the HRP One-Step Polymer is applied and incubated. Any excess of unbound polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
100 mL HRP One-Step-Polymer anti-Mouse/Rabbit (ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB High contrast kit. Materials required but not supplied: Positive and negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. HRP One-Step Polymer anti-Mouse/Rabbit 30 min. 8. Washing with wash buffer 3 x 2 min. 9. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: aqueous with AEC, permanent with DAB
Expected results:
During the reaction of the substrate with horseradish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horseradish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution can result in decreasing signal intensity. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 950 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
Anaplastic lymphoma kinase (ALK) is a novel receptor protein-tyrosine kinase. ALK can create a fusion protein with a nuclear protein gene called nucleophosmin (NPM) via the amino terminus of NPM and the catalytic domain of ALK. The product of this fusion protein is oncogenic.1 Studies have found this chromosomal translocation in most anaplastic large-cell non-Hodgkin's lymphomas, making ALK a good marker for anaplastic large cell lymphomas
Antibody Isotype:
IgG1-k
Monosan Range:
MONOSAN
Clone:
ALK-1
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Acute humoral rejection is mediated by antibodies to the donor endothelium that activate the classical complement pathway. This leads to a number of split products of complement proteins. C4d is a fragment of C 4 complement released during activation of the classic complement pathway by the antigen antibody complex. C4d deposits in peritubular capillaries and is regarded as an indirect sign of an antibody response. C4d can be a useful tool for indicating acute renal allograft rejection
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
SP91
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Jianghua C, et al. Clin Transplant. 2005; 19:785-91
References 2:
Kayler LK, et al. Transplantation. 2008; 85:813-20
References 3:
Ranjan P, et al. Nephrol Dial Transplant .2008; 23:1735-41
References 4:
Seemayer CA, et al. Nephrol Dial Transplant. 2007; 22:568-76
References 5:
Bouron-Dal Soglio D, et al. Hum Pathol. 2008; 39:1103-10
T-cell leukemia/lymphoma protein 1A (TCL1) is a member of theTCL1 family and enhances the phosphorylation and activation ofAKT1, AKT2 and AKT3. TCL1promotes the nuclear translocation ofAKT1 and enhances cell proliferation, stabilizes mitochondrial membrane potential and promotes cell survival. The expression of TCL1 is restricted to lymphoid cells. It is expressed early in lymphocyte differentiation. Strong expression ofTCL1 is found in a subset of mantle zone B lymphocytes and is expressed to a lesser extent by follicle center cells. In B cell neoplasia, TCL1 immunoreactivity is found in the majority of B cell lymphomas including lymphoblastic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, follicular lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma (60%), and primary cutaneous B cell lymphoma (55%). The expression of theTCL1genecharacterizes low-grade B cell lymphomas.
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
EP105
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Laine J, et al. Mol Cell2000, 6:395-407
References 2:
Pekarsky Y, et al. Proc Natl Acad Sci U S A, 2000, 97:3028-3033
References 3:
Narducci MG, et al. Cancer Res, 2000, 60:2095-2100
Glypican-3 (GPC3) is a membrane-bound heparin sulfate proteoglycan known to participate in cell growth and differentiation. GPC3 expression has been observed in the majority of hepatocellular carcinomas (HCC), but is rarely expressed in non-neoplastic hepatic tissue, making it a useful marker for HCC. Additionally, this marker is expressed in many yolk sac tumors.
Antibody Isotype:
IgG1
Monosan Range:
MONOSAN
Clone:
1G12
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Kandil DH, et al. Adv Anat Pathol. 2009; 16:125-9
References 2:
Coston WMP, et al. Am J Surg Pathol. 2008; 32:433-44
References 3:
Capurro M, et al. Gastroenterology. 2003; 125:89-97
References 4:
Zynger DL, et al. Am J Surg Pathol. 2006; 30:1570-5
Polyhisitidine tagged proteins (N- or C-terminus label)
Concentration:
1mg/ml
Conjugate:
Europium
Form:
Liquid
Purification:
Affinity Purified Using solid phase 6x Histidine IgG
Buffer:
50 mM tris (pH 7.8), 0.9% NaCl, 0.05% NaN3,
Storage:
-20C or colder
Disclaimer:
For in vitro Laboratory Use Only. Not for diagnostic or therapeutic use. Not for human or animal consumption. Suggested applications of our products are not recommendations to use our products in violation of any patent or as a license under any patent of ImmunoReagents, Inc. Product may not be resold or modified for resale without prior written approval of ImmunoReagents, Inc.
For in vitro Laboratory Use Only. Not for diagnostic or therapeutic use. Not for human or animal consumption. Suggested applications of our products are not recommendations to use our products in violation of any patent or as a license under any patent of ImmunoReagents, Inc. Product may not be resold or modified for resale without prior written approval of ImmunoReagents, Inc.
Human influenza hemagglutinin (HA) tagged proteins
Concentration:
Europium
Form:
Liquid
Purification:
Affinity Purified Using solid phase HA
Buffer:
50 mM tris (pH 7.8), 0.9% NaCl, 0.05% NaN3,
Storage:
-20C or colder
Disclaimer:
For in vitro Laboratory Use Only. Not for diagnostic or therapeutic use. Not for human or animal consumption. Suggested applications of our products are not recommendations to use our products in violation of any patent or as a license under any patent of ImmunoReagents, Inc. Product may not be resold or modified for resale without prior written approval of ImmunoReagents, Inc.
For in vitro Laboratory Use Only. Not for diagnostic or therapeutic use. Not for human or animal consumption. Suggested applications of our products are not recommendations to use our products in violation of any patent or as a license under any patent of ImmunoReagents, Inc. Product may not be resold or modified for resale without prior written approval of ImmunoReagents, Inc.
For in vitro Laboratory Use Only. Not for diagnostic or therapeutic use. Not for human or animal consumption. Suggested applications of our products are not recommendations to use our products in violation of any patent or as a license under any patent of ImmunoReagents, Inc. Product may not be resold or modified for resale without prior written approval of ImmunoReagents, Inc.
CD23 antigen is a 45-60 kDa membrane glycoprotein identified as a low affinity receptor for IgE production as well as a receptor for lymphocyte growth factor. CD23 is found in some mature B-cell lymphomas and in Reed-Sternberg cells in Hodgkin disease. Follicular dendritic cells and some activated B-cells within germinal centers express CD23 in high density and mantle zone B-cells are stained. The majority of chronic lymphocytic leukemias/small lymphocytic lymphomas are anti-CD23 positive, whereas mantle cell lymphomas are generally negative, so this marker is useful when applied with other markers to separate the small cell lymphomas.1,3 Precursor B and T lymphomas, myeloid neoplasms, and mature T-cell lymphomas are CD23 negative and other small cell lymphomas are occasionally positive. Anti-CD23 is expressed in activated mature B-cells expressing IgM or IgD, monocytes/macrophages, follicular dendritic cells, T-cell subsets, eosinophils, Langerhans cells and small lymphocytic lymphoma/chronic lymphocytic leukemia.
Antibody Isotype:
IgG1-k
Monosan Range:
MONOSAN
Clone:
1B12
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Kaiserlian, D, et al., Immunology 1993;80:90-95
References 2:
Aubry, JP et al., Oxford Univ Press- Oxford, NY, Tokyo 1987:417-419
References 3:
Pallesen G, Oxford Univ Press-Oxford, NY, Tokyo 1987:383-386
References 4:
Pezzella F et al. Br j Haematol. 2000 Feb;108(2): 369-76
References 5:
Kurtin PJ et al. Am J Clin Pathol. 1999 Sep;112(3): 319-29
Synthetic peptide derived from the carboxy terminal region of the human CD8 alpha chain coupled to a N-terminal cysteine, with the sequence C-KSDGKPSLSARYV. The peptide was coupled to bovine serum albumin and keyhole limpet hemocyanin.
Mouse anti Human CD8 antibody, clone 4B11 recognizes the human CD8 cell surface antigen, a ~32 kDa glycoprotein expressed by the cytotoxic/suppressor subset of T-cells and weakly by NK cells. Mouse anti Human CD8 antibody, clone 4B11 was raised based on an earlier successful strategy used to generate rabbit polyclonal antibodies against human CD8 employing the same immunizing peptide (Mason et al. 1992).Mouse anti Human CD8 antibody, clone 4B11 has been reported as being suitable for use in Western blotting (Williamson et al. 1998) .
PU.1 is a transcription factor that has been shown to be important for normal B-cell development. PU.1 belongs to the ETS family of transcription factors. It is expressed in the myeloid lineage and in immature as well as mature B-lymphocytes, with the exception of plasma cells. PU.1 is essential during early B-cell differentiation. The absence of PU.1 results in total block of B-cell development at the prepro stage. Very little is known about PU.1 function in later stages of B-cell development. PU.1 does not seem to play a role in the end-stage of B-cell development and is not expressed in plasma cells. PU.1 exerts an important role in the regulation of the expression of crucial B-cell proteins, such as immunoglobulin (Ig) genes, and CD20 and its putative binding sites were also identified in the promoters of CD79, CD10, and CD22. PU.1 binds to the 3 enhancer region of both the Ig kappa and lambda light chain genes and it also regulates the immunoglobulin heavy chain genes through the intron enhancer region.
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
EPR3158Y
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Recognizes a protein of 150kDa, which is identified as the high molecular weight variant of Caldesmon. Two closely related variants of human caldesmon have been identified which are different in their electrophoretic mobility and cellular distribution. The h-caldesmon variant (120150kDa) is predominantly expressed in smooth muscle whereas l-caldesmon (7080kDa) is found in non- muscle tissue and cells. Neither of the two variants has been detected in skeletal muscle. This MAb recognizes only the 150kDa variant (h-caldesmon) in Western blots of human aortic media extracts and is unreactive with fibroblast extracts from cultivated human foreskin. Caldesmon is a developmentally regulated protein involved in smooth muscle and non-muscle contraction. Pretreatment: Heat induced epitope retrieval in 10 mM citrate buffer, pH6.0, for 20 minutes is required for IHC staining on formalin-fixed, paraffin embedded tissue sections. Note: Dilution of the antibody in 10% normal goat serum followed by a goat anti-mouse secondary antibody-based detection is recommended. Control tissue Uterus, blood cessels, smooth muscle or leiomyosarcoma. Staining Cytoplasmic
Mouse anti human CD236 antibody, clone HEA-125 recognizes CD326 also known as Adenocarcinoma-associated antigen, Epithelial glycoprotein 314 or Epithelial Cell Adhesion Molecule (Ep-CAM). CD326 is a 314 amino acid ~34 kDa single pass type I transmembrane glycoprotein nearing a single thyroglobulin type-1 domain (UniProt: P16422).CD326 is expressed on the basolateral membrane of cells by the majority of epithelial tissues, with the exception of adult squamous epithelium and some specific epithelial cell types including hepatocytes and gastric epithelial cells.CD326 expression has been reported to be a possible marker of early malignancy, with expression being increased in tumour cells (Balzar et al. 1999), and de novo expression being seen in dysplastic squamous epithelium (Winter et al. 2003). CD326 has been identified independently by a number of groups, and it has been known by a variety of names including Epithelial Specific Antigen, MOC31 (Proca et al. 2000) and Ber-EP4 (Patriarca et al. 2012).
The PAX-5 gene is essential for B-cell differentiation. There are at least four isoforms, of which PAX-5a has been most studied. PAX-5 encodes the 50 kDa B-cell specific activator protein, BSAP. PAX-5 is expressed by pro-, pre-and mature B-cells, but is downregulated during terminal differentiation of plasma cells. PAX-5 influences the expression of other B-cell specific genes, including CD19 and CD20 and CD79a, preceding the expression of CD20. PAX-5 is silenced at the plasma cell stage under the influence of B-lymphocyte-induced maturation protein-1 (PRDM1). PAX-5 is expressed during mouse embryogenesis within the developing brain in a way that is temporarily and spatially tightly condoled. PAX-5 deficient mice show deformation of the mid-brain. Expression in human embryogenesis occurs in the mesoencephalon and spinal cord. Pretreatment: Heat induced epitope retrieval in 10 mM citrate buffer, pH6.0, for 20 minutes is required for IHC staining on formalin-fixed, paraffin embedded tissue sections. (dilution 1:100 - 1:200) The optimal dilution for a specific application should be determined by the investigator. Note: Dilution of the antibody in 10% normal goat serum followed by a goat anti-mouse secondary antibody-based detection is recommended. Control tissue Tonsil; staining nuclear
Permanent AP Red Kit is developed for immunohistochemical and in situ-hybridisation staining procedures with alkaline phosphatase. Permanent AP Red leads to the formation of a magenta-red precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light or fluorescence microscopy.
500 ml Permanent AP Red Buffer 8 ml Permanent AP Red Chromogen 1 Dilution Vial
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution prepared is stable for about 60 minutes and should therefore be used directly after preparation. Excess working solution should be discarded. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation:
Reagent preparation (Preparation of the working solution) 1) Pipette 2.5 ml AP Red Buffer into the provided dilution vial and let it come to room temperature. The chromogen should still be kept cool. 2) Directly prior to use add 1 drop of Permanent AP Red Chromogen into the buffer. Mix thoroughly. 3) The solution is stable for about 60 minutes. Preparation should be done directly before use. Make sure to pipet the chromogen/substrate mix on the last slide of the staining run within 40 min after mixing. If you want to prepare other quantities of the working solution, please use same ratio AP Red Buffer and Chromogen
Procedure:
1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply freshly prepared Permanent AP Red working solution onto the slide. Incubate for 10 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium. Note: It is also possible to mount Permanent AP Red with aqueous mounting media.
Expected results:
During the reaction of the substrate with alkaline phosphatase in presence of the chromogen Permanent AP Red, a magenta-red precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light or fluorescence microscopy (Texas Red filter).
Trouble shooting:
If you observe unusual staining or other deviations from the expected results please read these instructions carefully, or contact our technical support. Also refer to the instructions of the detection systems for guidance on general troubleshooting.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining. Please refer to the instructions of the detection system for guidance on general quality control procedures.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). In some tissues endogenous alkaline phosphatase activity may cause non-specific staining. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Therefore, tissues of this origin should be stained with peroxidase detection systems. A higher sensitivity can be obtained when a second chromogenic substrate step is used (i. e. 2 x 10 min Permanent AP Red). Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. A longer exposure to absolute ethanol can result in decreasing staining intensity. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid contact of reagents or specimen with eye, skin or mucous membrane. In case of a reagent or specimen coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. A material safety data sheet (MSDS) is available upon request.
CD33, also known as gp67 or SIGLEC-3, is a 67 kDa glycosylated transmembrane protein that is a member of the sialic acid-binding immunoglobulin-like lectin (siglec) family. Although the precise physiological function of CD33 is unknown, it may mediate cell to cell adhesion and modulate inflammatory and immune response. In normal tissue, anti-CD33 labels myeloid cells (especially myeloid precursors), liver Kupffer cells, lung alveolar macrophages, and placental syncytiotrophoblasts. In neoplastic tissue, anti-CD33 is useful for the identification of acute myeloid leukemia.
Antibody Isotype:
IgG2b
Monosan Range:
MONOSAN
Clone:
PWS44
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Freeman SD, et al. Blood. 1995; 85:2005-12
References 2:
Laszlo GS, et al. Oncotarget. 2016; 7:43281-94
References 3:
Crocker, PR et al. Biochem Soc Symp 2002; 69:83-96
CD4 is a 55 kD glycoprotein expressed on the surface of T-helper/regulatory T-cells, monocytes, macrophages, and dendritic cells. Anti-CD4 is used in the immune phenotyping of lymphoproliferative disorders. The majority of peripheral T-cell lymphomas are derived from the T-helper/regulatory cell subset so that most mature T-cell neoplasms are CD4+ CD8-. As with other T-cell antigens, CD4 may be aberrantly expressed in neoplastic T-cells so that the evaluation of such tumors requires the application of a panel of markers in order to identify tumors with CD4 aberrant expression.
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
1F6
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Leong AS-Y, et al. Greenwich Medical Media Ltd. 2003
References 2:
Akiyama T, et al. Pathol Int. 2008; 58:626-34
References 3:
Garcia-Herrera A, et al. J Clin Oncol. 2008; 26:3364-71
Recognizes a protein of 67kDa, which is identified as estrogen receptor (ER) alpha. The ER gene consists of more than 140kb of genomic DNA divided into 8 exons, being translated into a protein with six functionally discrete domains, labeled A through F. This antibody strongly stains the nucleus of epithelial cells in breast carcinomas. The ER is an important regulator of growth and differentiation in the mammary gland. Presence of ER in breast tumors indicates an increased likelihood of response to antiestrogen (e.g. tamoxifen) therapy. Pretreatment: Heat induced epitope retrieval in 10 mM citrate buffer, pH6.0, or in 50 mM Tris buffer pH9.5, for 20 minutes is required for IHC staining on formalin-fixed, paraffin embedded tissue sections. Note: Dilution of the antibody in 10% normal goat serum followed by a goat anti-rabbit secondary antibody-based detection is recommended. Control tissue Breast carcinoma. Staining nuclear.
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
ZR2
Concentration:
n/a
Format:
Concentrate
Storage buffer:
Tissue culture supernatant with 0.2% BSA and 15mM sodium azide
Oct-4 is a transcription factor that functions in the regulation and maintenance of pluripotency in embryonic stem and primordial germ cells. Oct-4 immunoreactivity has been demonstrated in gonadal and extra-gonadal seminomas, dysgerminomas and embryonal carcinomas. In addition, the immunohistochemical detection of Oct-4 assists in the evaluation of intratubular germ cell neoplasia (IGCN).
Antibody Isotype:
IgG1-k
Monosan Range:
MONOSAN
Clone:
MRQ-10
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Cheng L, et al. J Pathol. 2007; 211:1-9
References 2:
Weissferdt A, et al. Hum Pathol. 2015; 46:376-83
References 3:
Browne P, et al. Am J Clin Pathol. 2003 Nov;120(5):767-77
References 4:
García-Cosío M, et al. Mod Pathol. 2004 Dec;17(12):1531-8
References 5:
Gibson SE, et al. Am J Clin Pathol. 2006 Dec;126(6):916-24
The Plus AP Polymer anti-Mouse is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with monoclonal primary antibodies obtained from mouse. The reagent can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Polymer anti-Mouse is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The reagent is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mouse. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus AP Polymer anti-Mouse avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the AP polymer is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the AP-polymer is applied and incubated. Any excess of unbound AP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Fast Red leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red), New Fuchsin (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
100 ml AP-Polymer anti-Mouse (Ready-to-use) Substrate systems recommended: Permanent AP Red kit, Fast Red substrate kit, New Fuchsin kit. Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out
Procedure:
1. Blocking Solution (protein block, this step is optional) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 5 min. 5. AP-polymer anti Mouse 30 min. 6. Washing with wash buffer 3 x 2 min. 7. Fast Red, Permanent AP Red, NBT/BCIP or New Fuchsin 5-15 min. (Controlling the colour intensity via light microscope is recommended.) 8. Stopping the reaction with distilled H2O when the desired colour intensity is attained 9. Counterstaining and blueing 10. Mounting: aqueous with Fast Red, permanent with Permanent AP Red, NBT/BCIP or New Fuchsin
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use a Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high).
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light.Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of the reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagent must be avoided, since otherwise non-specific staining might appear. ProClin 950 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
Smoothelin is a constituent of the smooth muscle cell cytoskeleton protein exclusively found in differentiated smooth muscle cells (SMC). Cells with SMC-like characteristics, such as myofibroblasts and myoepithelial cells, as well as skeletal and cardiac muscle do not contain smoothelin.1,2 To distinguish bladder muscularis mucosae (MM) from muscularis propria (MP) muscle bundles is crucial for accurate staging of bladder carcinoma. Strong smoothelin expression is nearly exclusively observed in muscularis propria. Therefore, the staining pattern of MP (strongly positive) and MM (negative or weakly positive) makes this technique an attractive diagnostic tool for the sometimes difficult task of staging bladder urothelial carcinoma, such as in transurethral resection specimens of urinary bladder tumors.3-8 Differentiating between smooth muscle tumors and other mesenchymal neoplasms of the GI tract can be challenging in small biopsies.
Antibody Isotype:
IgG1
Monosan Range:
MONOSAN
Clone:
R4A
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
van der Loop, FT et al. J Cell Biol 1996; 134:401-411
CD19 is a glycoprotein on the surface of mature B cells, it works in conjunction with receptors and proteins to regulate B-cell signaling. CD19 is present in both normal and malignant B cells, and hence being valuable for the identification of B-cell neoplasms such as diffuse large B-cell lymphoma.
Antibody Isotype:
IgG1-k
Monosan Range:
MONOSAN
Clone:
MRQ-36
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Steinmetz OM, et al. Transplantation. 2007 15;84(7):842-50
References 2:
Teng YK, et al. Arthritis Rheum. 2007 ;56(12):3909-18
The progesterone receptor (PgR) is an estrogen regulated protein. It has been proposed that expression of PgR determination indicates a responsive estrogen receptor (ER) pathway, and therefore, may predict likely response to endocrine therapy in human breast cancer. A number of studies have shown that PgR determination provides supplementary information to ER, both in predicting response to endocrine therapy and estimating survival. PgR has proved superior to ER as a prognostic indicator in some studies. Pretreatment: Heat induced epitope retrieval in 10 mM citrate buffer, pH6.0 for 20 minutes is required for IHC staining on formalin-fixed, paraffin embedded tissue sections. Note: Dilution of the antibody in 10% normal goat serum followed by a goat anti-rabbit secondary antibody-based detection is recommended. Control tisse Breast, breast ducatl carcinoma. Staining Nuclear
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
ZR4
Concentration:
n/a
Format:
Concentrate
Storage buffer:
Tissue culture supernatant with 0.2% BSA and 15mM sodium azide
The Plus HRP Polymer Kit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Polymer Kit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of horse radish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP Polymer Kit avoids the problem of background staining caused by endogenous biotin in the tissue
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRPpolymer is minimized by incubation with a protein blocking solution (Blocking Solution, provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the enhancement reagent (PostBlock) is applied and incubated. A second washing is followed by the application of the HRP-polymer. Any excess of unbound HRP-polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
100 ml Blocking Solution Reagent 1 (ready-to-use) 100 ml PostBlock Reagent 2 (ready-to-use) 100 ml HRP-Polymer (Mouse/Rabbit) Reagent 3 (ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB High contrast kit. Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. ? Deparaffinise and rehydrate paraffin-embedded tissue sections. ? Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. ? Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. PostBlock (Reagent 2, yellow) 20 min. 8. Washing with wash buffer 3 x 5 min. 9. HRP-polymer (Reagent 3, red) 30 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. We guarantee that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin is used for stabilisation. A material safety data sheet is available upon request.
The Plus AP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus AP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kits, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Broad Spectrum is polyvalent. With this kit it is therefore possible to detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized via incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP110) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 125 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion.Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP110 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (substrate buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 12. Wash with distilled H2O 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolized by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see also Limitations of the procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3), used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
GATA binding protein 3 or GATA3, is a zinc finger transcription factor and plays an important role in promoting and directing cell proliferation, development, and differentiation in many tissues and cell types.1 The human GATA3 gene has been mapped to chromosome 10p15.3 GATA3 expression is primarily seen in breast carcinoma and urothelial carcinoma. Anti-GATA3 can also be useful in the identification of unknown primary carcinoma when carcinomas of the breast or bladder are a possibility
Antibody Isotype:
IgG1-k
Monosan Range:
MONOSAN
Clone:
L50-823
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Higgins JP, et al. Am J Surg Pathol. 2007; 31:673-80
The outer light-harvesting antenna of the photosystems (PSI and PSII) of the green unicellular alga Chlamydomonas reinhardtii is composed of pigment-binding proteins belonging to the Lhc family highly conserved in photosynthetic eukaryotes. Some of the Lhc-subtypes of Chlamydomonas share sufficient high similarity with the respective functional homologs in plants to allow specific detection with antisera generated against conserved peptide domains from plant Lhc-proteins.
Product Type:
Antibody
Antibody Type:
Polyclonal
Format:
Lyophilized
Storage Temp:
Store lyophilized/reconstituted at -20 °C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
Lhcb4 - higher plants (use AS04 045 for those organsims), algae, cyanobacteria
Selected references:
Lhcb2 Du et al. (2018). Galactoglycerolipid Lipase PGD1 Is Involved in Thylakoid Membrane Remodeling in Response to Adverse Environmental Conditions in Chlamydomonas. Plant Cell. 2018 Feb;30(2):447-465. doi: 10.1105/tpc.17.00446. Lhcb4Jeong et al. (2017). Deletion of the chloroplast LTD protein impedes LHCI import and PSI-LHCI assembly in Chlamydomonas reinhardtii. J Exp Bot. 2017 Dec 30. doi: 10.1093/jxb/erx457. Lhcb5 and Lhcbm5 Takahashi et al. (2006). Identification of the mobile light-harvesting complex II polypeptides for state transitions in Chlamydomonas reinhardtii. PNAS 103:477-482
Human prostein is a 553 aa protein identified by cDNA library substraction abd subsequent highthroughput microarray ascreening of prostate cancer. Prostein has multiple transmemberane domains. Prostein has been shown to be uniquely expressed in normal and cancerous prostatic tissues. By immunohistochemistry, prostein is expressed in the vast majority of normal and malignant prostatic tissues, regardless of grade and metastatic status. No protein expression is detected in normal and malignant tissue samples representing the great majority of essential tissues and tumors. Prostein is expressed in most of poorly differentiated prostatic carcinoma, including small cell prostate carcinoma. Prostein is more specific and sensitive for prostatic carcinomas than PSA and PSAP. Pretreatment: Heat induced epitope retrieval in 10 mM citrate buffer, pH6.0, for 20 minutes is required for IHC staining on formalin-fixed, paraffin embedded tissue sections. Note: Dilution of the antibody in 10% normal goat serum followed by a goat anti-rabbit secondary antibody-based detection is recommended. Control tissue Normal prostate or postate carcinoma. Staining cytoplasmic
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
ZR9
Concentration:
n/a
Format:
Concentrate
Storage buffer:
Tissue culture supernatant with 0.2% BSA and 15mM sodium azide
Originally, the 2G9 monoclonal antibody (mAb) was described as identifying a 15 kDa protein found in the cytoplasmic granules of cytotoxic T cells that might be part of a larger 40 kDa molecule, ubiquitously expressed, named p40-TIA-1 and often referred to as TIA-1 in the literature (1, 2). Now, however, there is evidence that the 2G9 mAb identifies a 17 kDa cytoplasmic granule membrane protein named GMP-17 that has no similarity with p40-TIA-1 (3). The GMP-17 antigen is a 165 amino acid protein with 4 transmembrane domains: but it is not a typical member of the fourtransmembrane superfamily. It is identical with previously identified cytotoxic granule proteins called NKG7 and GIG-1 for GCSF induced gene protein 1 , isolated from NK cells and granulocyte-colony-stimulatingfactor- treated mononuclear cells, respectively (4, 5). Pretreatment: Heat induced epitope retrieval in 10 mM citrate buffer, pH6.0, for 20 minutes is required for IHC staining on formalin-fixed, paraffin embedded tissue sections. Note: Dilution of the antibody in 10% normal goat serum followed by a goat anti-mouse secondary antibody-based detection is recommended. Control tissue Tonsil. Staining granular
Antibody Isotype:
IgG1
Monosan Range:
MONOSAN
Clone:
2G9A10F5
Concentration:
n/a
Format:
Purified
Storage buffer:
Liquid purified Ascites, purified with Protein G Chromatography with 15mM Sodium Azide
Storage:
2-8°C
References 1:
Anderson, P. et al, 1990, J. Immunol., 2, 144, 574.
References 2:
Tian, Q., et al, 1991, Cell, 67, 629-639.
References 3:
Medley, et al, 1996, Proc. Natl. Acad. Sci. U S A, 93, 2, 685-9.
References 4:
Turman, M.A., et al, 1993, Hum. Immunol., 36, 1, 34-40.
References 5:
Shimane, et al, 1994, Biochem Biophys Res Commun., 199, 1, 26-32.
The oncogenic transcription factor, c-MYC, has a crucial role in growth control, differentiation, cellular metabolism and apoptosis and is associated with variety of tumors. MON 3393 stains this protein in tissues from colorectal adenocarcinoma, breast invasive ductal carcinoma, prostate adenocarcinoma, Burkitt lymphoma, and diffused large B-cell lymphoma.
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
EP121
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Cyclins are proteins that govern transitions through distinct phases of the cell cycle by regulating the activity of the cyclin-dependent kinases. Cyclin D1, one of the key cell cycle regulators, is a putative proto-oncogene found in a wide variety of human neoplasms. This antibody is useful for distinguishing mantle cell lymphomas (cyclin D1 positive).
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
SP4
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Aagaard, L, et al. International J of Cancer 1995;61(1):115-120
References 2:
Bartkova, J, et al. Cancer Research 1995;55:949-956
References 3:
Bartkova, J, et al. Oncogene 199510(4):775-778
References 4:
Bartkova, J, et al. J of Pathology 1994;172(3):237-245
References 5:
Lukas, J, et al. Molecular and Cellular Biology 1995;15(5):2600-2611
Ochratoxin A, a toxin produced by Aspergillus ochraceus and Penicillium verrucosum, is one of the most abundant food-contaminating mycotoxins in the world. The toxin has been found in the tissues and organs of animals, including human blood and breast milk. Ochratoxin A can cause immunosuppression and immunotoxicity in animals.
The antibody reacts with progesterone receptor forms alpha and beta. This antibody stains nuclei in breast, ovarian and endometrial epithelia, as well as myometrial nuclei. Since the early 1990s the immunohistochemical (IHC) assay determination of progesterone receptor status has replaced the dextran-coated charcoal method as a prognostic indicator in breast carcinoma. IHC has shown to be superior in prognostic significance when using any one of several available methods of quantitation using this technique.
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
Y85
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Dabbs D. Diagnostic Immunohistochemistry, 2nd edition. p 728-32
References 2:
Dunnwald LK, et al. Breast Cancer Res. 2007;9(1):R6
References 3:
Leong A S-Y, et al. Manual of diagnostic immunohistochemistry, 2nd edition. p 375-76
Agrisera
AS05 070set
50 µl / tube of each primary antibody. 100 µl / tube of each protein standard. 20 µl (2 x 10 µl) of secondary antibody. 10 ml trial pack of ECL reagent.
Store lyophilized/reconstituted at -20 °C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
Oct-binding factor-1 (OBF1), also known as BOB.1, is a B-cell-specific coactivator which has been mapped to chromosome 11q23. Expression of BOB.1/OBF.1 is restricted largely to mature B-cells, with germinal center B-cells normally staining for BOB.1.2,3 Analyses of BOB.1/OBF.1 expression in a variety of established B-cell lines representing different stages of B-cell development has suggested a constitutive, B-cell-specific expression pattern. LP cells in nodular lymphocyte predominant Hodgkin lymphoma, because they are germinal center-derived, are consistently immunoreactive for BOB.1. Conversely, only some cases of classical Hodgkin lymphoma show BOB.1 immunoreactivity within the Hodgkin and Reed-Sternberg cells. Expression of BOB.1/OBF.1 has been reported in follicular center cell lymphoma, diffuse large B-cell lymphoma and some cases of acute myeloid leukemia. B-CLL, marginal zone lymphoma, and mantle cell lymphoma may show weak to moderate immunoreactivity.
Antibody Isotype:
IgG-1
Monosan Range:
MONOSAN
Clone:
SP92
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Junker S, et al. Genomics. 1996; 33:143-5
References 2:
Steimle-Grauer SA, et al. Virchows Arch. 2003; 442:284-93
Anti-myeloperoxidase detects granulocytes and monocytes in blood and precursors of granulocytes in the bone marrow. This antibody can detect myeloid cell populations of the bone marrow as well as in other sites.
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
SP72
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Pinkus GS, et al. Mod Pathol. 1991; 4:733-41
References 2:
Chang CC, et al. Am J Clin Pathol. 2000; 114:807-11
References 3:
Kaleem Z, et al. Am J Clin Pathol. 2001; 115:876-84
References 4:
Audouin J, et al. Int J Surg Pathol. 2003; 11:271-82
PAX-8 is a transcription factor expressed during embryonic development of Müllerian organs, kidney, and thyroid, with continued expression in some epithelial cell types of these mature tissues.1 It can be useful for marking several types of carcinoma including ovarian serous carcinoma, clear cell renal cell carcinoma, and papillary thyroid carcinoma.1-5 Additionally, PAX-8 is not found in the epithelial cells of the breast, lung, mesothelium, stomach, colon, pancreas and other sites.
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
MRQ-50
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Ozcan A, et al. Mod Pathol. 2011; 24:751-64
References 2:
Laury AR, et al. Am J Surg Pathol. 2011; 35:816-26
References 3:
Nonaka, D et al. Am J Surg Pathol. 2008; 32:1566-71
Anti-CD5 is a pan T-cell marker that also reacts with a range of neoplastic B-cells. CD5 expression is useful in distinguishing mature T-cell neoplasms and differentiating among mature small lymphoid cell malignancies. Anti-CD5 does not react with granulocytes or monocytes.
Antibody Isotype:
IgG1
Monosan Range:
MONOSAN
Clone:
SP19
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Jones NH, et al., Nature 1986;323: 346-349
References 2:
Tan SH et al. Br J Dermatol. 2003 Sep;149(3): 542-53
References 3:
Chang CC et al. Mod Pathol. 2002 Oct;15(10): 1051-7
References 4:
Hatano B et al. Pathol Int. 2002 May-Jun;52(5-6): 400-5
References 5:
West RB et al. Am J Clin Pathol. 2002 Apr;117(4): 636-43
NPR1 (regulatory protein NPR1) is a key positive regulator of the SA-dependent signaling pathway that negatively regulates JA-dependent signaling pathway. Controls the onset of systemic acquired resistance (SAR). Subcellular localization: cytoplasm, nucleaus (following induction by salicylic acid treatment or after pathogen infection. Alternative names: BTB/POZ domain-containing protein NPR1, Non-inducible immunity protein 1, Nim1, Salicylic acid insensitive 1, Sai1, ATNPR1.
Product Type:
Antibody
Antibody Type:
Polyclonal
Format:
Lyophilized
Storage Temp:
Store lyophilized/reconstituted at -20 °C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles,Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
Host Animal:
Rabbit
Species Reactivity:
Arabidopsis thaliana
Expected Species:
Arabidopsis thaliana
Immunogen:
KLH-conjugated peptide, chosen from NPR1 sequence of Arabidopsis thaliana, TAIR: AT1G64280, UniProt: P93002
For successful detection using NPR1 antibody please follow protocol suggested below. NPR1 protein readily oligomerizes, in addition to any naturally occurring oligomer, during extraction. Therefore 50 mM DTT has to be used as well as denaturation at 75 C for 15 minutes. Engogenous NPR1 level is very low, thereofre SA treatment is absolutely necessary for good detection.This antibody is recognizing NPR1-GFP in the 35S overexpression line.
DOG1 is a calcium-dependent chloride channel protein that is encoded by a gene called TMEM16A (TMEM16 FLJ10261, ANO1, ORAOV2, and AOS2) located on chromosome 11q13. DOG1 has many significant functions such as regulation of the cholinergic activity of gastrointestinal smooth muscle 2 and regulation of both the survival and proliferation of cells. Anti-DOG1 antibody has been shown to be useful in the identification of gastrointestinal stromal tumors (GIST).
Antibody Isotype:
IgG
Monosan Range:
MONOSAN
Clone:
SP31
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Kang HG, et al. Mod Pathol. 2011; 24:866-77
References 2:
Rizzo FM, et al. BMC Cancer. 2016; 16:87
References 3:
Katoh M, et al.Int J Oncol. 2003; 22:1375-81
References 4:
Stanich JE, et al. Am J Physiol Gastrointest Liver Physiol. 2011; 1044-51
The Plus AP Polymer Kit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Polymer Kit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer in this kit consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice and rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus AP Polymer Kit avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the AP polymer is minimized by incubation with a protein blocking solution (Blocking Solution, provided with this kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the enhancement reagent (PostBlock) is applied and incubated. A second washing is followed by the application of the AP-polymer. Any excess of unbound APpolymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent AP Red leads to the formation of a magentared product of reaction at the place of the target antigen.
Reagents provided:
100 ml Blocking Solution Reagent 1 (ready-to-use) 100 ml PostBlock Reagent 2 (ready-to-use) 100 ml AP-Polymer (Mouse/Rabbit) Reagent 3 (ready-to-use) Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 5 min. 5. PostBlock (Reagent 2, yellow) 20 min. 6. Washing with wash buffer 3 x 5 min. 7. AP-polymer (Reagent 3, red) 30 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent AP Red 10-20 min. (Controlling the colour intensity via light microscope is recommended.) 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: permanent or aqueous with Permanent AP Red
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure).
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Therefore, tissues of this origin should be stained with peroxidase detection systems (i.e. POLHRP-125). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the BlockingSolution instead of just draining it away as in other procedures. We will guarantee that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and ProClin 950 used for stabilisation. Material safety data sheets (MSDS) are available upon request.
Uroplakins (UPs) are a family of transmembrane proteins (UPs Ia, Ib, II and III) that are specific differentiation products of urothelial cells. In non-neoplastic mammalian urothelium, UPs are expressed in the luminal surface plasmalemma of superficial (umbrella) cells, forming complexes of 16nm crystalline particles. UPIII is specific for tumors of urothelial origin and, when used in combination with other markers, can aid in the diagnosis of primary and metastatic tumors.
Antibody Isotype:
IgG1
Monosan Range:
MONOSAN
Clone:
AU-1
Concentration:
n/a
Storage buffer:
Tris Buffer, pH 7.3-7.7, containing 1% BSA and <0.1% Sodium Azide
Storage:
2-8°C
References 1:
Moll R, et al. Am J Pathol. 1995; 147:1383-97
References 2:
Olsburgh J, et al. J Pathol. 2003; 199:41-9
References 3:
Parker DC, et al. Am J Surg Pathol. 2003; 27:1-10
References 4:
Ohtsuka Y, et al. BJU Int. 2006; 97:1322-6
References 5:
Logani S, et al. Am J Surg Pathol. 2003 Nov;27(11):1434-41
The Fast AP One-Step Polymer anti-Mouse/Rabbit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It was developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE. It is intended for in vitro diagnostic use.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Fast AP One-Step Polymer anti-Mouse/Rabbit is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer consists of several molecules of secondary antibodies covalently bound to several molecules of alkaline phosphatase (AP). Visualisation occurs via an enzymesubstrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. Cross-reactivity with primary antibodies from rat has been observed. In contrast to other detection techniques, which often use the streptavidin-biotin system the Fast AP One-Step Polymer anti-Mouse/Rabbit avoids the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the AP OneStep Polymer is minimized by incubation with a protein blocking solution. This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the AP One-Step Polymer is applied and incubated. Any excess of unbound polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent AP Red leads to the formation of a magentared product of reaction at the place of the target antigen.
Reagents provided:
100 ml Fast AP One-Step Polymer anti-Mouse/Rabbit (ready-to-use) Substrate systems recommended: Permanaent AP Red kit Materials required but not supplied Positive and negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS Blocking Solution (for protein blocking, optional) Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagent, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Blocking Solution (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 5 min. 5. AP One-Step Polymer anti-Mouse/Rabbit 30 Min. 6. Washing with wash buffer 3 x 2 min. 7. Permanent AP Red, 10-20 min. (Controlling the colour intensity via light microscope is recommended.) 8. Stopping the reaction with distilled H2O when the desired colour intensity is attained 9. Counterstaining and blueing 10. Mounting: permanent or aqueous with Permanent AP Red Kit
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high).
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
?-Casein is expressed as 13 genetic variants resulting from single nucleotide polymorphisms (SNP) in the CSN2 gene. The most frequent genetic variants in western dairy breeds are ?-Casein A1 and ?-Casein A2. The two types of ?-Casein protein, A1 and A2, differ by a single-point mutation at amino acid position 82 (P82/H82). The Biosensis Bovine A1 ?-Casein enzyme-linked immunosorbent assay (ELISA) Kit is a sandwich ELISA that allows the quantification of the bovine A1 ?-Casein isoform in 5 hours. This kit consists of a pre-coated rabbit anti-Bovine ?-Casein polyclonal capture antibody, a chicken anti-bovine A1 ?-Casein detection antibody and a horseradish peroxidase (HRP)-conjugated donkey anti-chicken IgY antibody. The addition of a substrate (3,3',5,5' -tetramethylbenzidine, TMB) yields a coloured reaction product which is directly proportional to the concentration of Bovine A1 ?-Casein present in samples and protein standards. Extensive validation has shown accurate quantification of A1 ?-Casein in UHT (Ultra High Temperature) treated milk or long life milk, organic pasteurized and homogenized full cream milk, fresh pasteurized and homogenized milk, cold-pressed raw milk (non-pasteurized, non-homogenized), and biodynamic full-cream whole milk. Please refer to the kit protocol for specific use instructions. The A2 isoform is not detected in this ELISA assay.
Product Type:
ELISA Assay
Species Reactivity:
Bovine
Immunogen:
Purified bovine beta Casein and peptide specific peptides for A1
Applications:
ELISA
Application Details:
ELISA. For the quantification of A1 Beta casein (A1) in Bovine Milk. Please download the detailed product insert for complete instructions for the successful use of this ELISA. Use only as directed.
Alternative Names:
CSN2; Bovine A1 Beta Casein; A1;
Biosensis Brand:
Biosensis®
Detection Method:
Colorimetric
Shelf Life:
12 months after date of receipt unopened.
Use:
For research use only.
Kit Components:
The ELISA kit box contains 96-well pre-coated strip plate(s), protein standards, detection reagents, wash and sample buffers, substrate buffer, stop solution and detailed protocols.
Specificity:
Bovine A1 beta-casein
Storage:
Store at 2-8°C
Range:
3.1 - 200 ng/mL
Sample Type:
Bovine Milk
Sensitivity:
This bovine A1 beta-casein ELISA kit detects a minimum of 3 ng/mL A1 beta-casein in assay buffer (defined as A1 concentration at blank OD plus 3x standard deviations of the blank OD (n=10))
Cross Reactivity:
No cross-reactivity is observed with bovine A2 beta-Casein
Factor VIIa (FVIIa) is a key serine protease involved in the initiation of the coagulation cascade. FVIIa requires tissue factor (TF), a membrane bound protein, as an essential cofactor for maximal activity towards its biological substrates Factor X, Factor IX and Factor VII (FVII).
Product Type:
Antibody
Antibody Type:
Polyclonal
Format:
Lyophilized
Storage Temp:
Store lyophilized/reconstituted at -20 °C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them, to avoid any losses that might occur from material adhering to the cap or sides of the tube.
Host Animal:
Rabbit
Species Reactivity:
Human
Expected Species:
Primates
Immunogen:
Recombinant human Factor VIIa (NovoSeven , Novo Nordisk A/S Denmark)
Applications:
Prothrombin assay with human plasma (PT), thrombin generation assay, inhibition assay with Factor VIIa with TF and a chromogenic substrate, inDirect ELISA (I-ELISA)
No confirmed exceptions from predicted reactivity are currently known
Selected references:
Lopez-Vilchez et al. (2009). Traffic of rFVIIa through Endothelial Cells and Redistribution into Subendothelium: Implications for a Prolonged Hemostatic Effect. Journal of Coagulation Disorders, October 1: (1). (immunolocalization)
Special application note:
No significant difference in binding to Factor VII and Factor VIIa.Antibody is purified to total IgG fraction, followed by purification on specific FVIIa column.
?-Casein is expressed as 13 genetic variants resulting from single nucleotide polymorphisms (SNP) in the CSN2 gene. The most frequent genetic variants in western dairy breeds are ?-Casein A1 and ?-Casein A2. The two types of ?-Casein protein, A1 and A2, differ by a single-point mutation at amino acid position 82 (P82/H82). The Biosensis Bovine A2 ?-Casein enzyme-linked immunosorbent assay (ELISA) Kit is a sandwich ELISA that allows the quantification of the bovine A2 ?-Casein isoform in 5 hours. This kit consists of a pre-coated rabbit anti-bovine ?-Casein polyclonal capture antibody, a chicken anti-bovine A2 ?-Casein detection antibody and a horseradish peroxidase (HRP)-conjugated donkey anti-chicken IgY antibody. The addition of a substrate (3,3',5,5' -tetramethylbenzidine, TMB) yields a coloured reaction product which is directly proportional to the concentration of Bovine A2 ?-Casein present in samples and protein standards. Extensive validation has shown accurate quantification of A2 ?-Casein in full cream milk, skim milk and reconstituted A2 milk powder. Please refer to the kit protocol for specific use instructions. The A1 isoform is not detected in this ELISA assay.
Product Type:
ELISA Assay
Species Reactivity:
Bovine
Immunogen:
Purified bovine beta Casein and peptide specific peptides for A2
Applications:
ELISA
Application Details:
ELISA. For the quantification of A2 Beta casein (A2) in Bovine Milk. Please download the detailed product insert for complete instructions for the successful use of this ELISA. Use only as directed.
Alternative Names:
CSN2; Bovine A2 Beta Casein; A2;
Biosensis Brand:
Biosensis®
Detection Method:
Colorimetric
Shelf Life:
12 months after date of receipt unopened.
Use:
For research use only.
Kit Components:
The ELISA kit box contains 96-well pre-coated strip plate(s), protein standards, detection reagents, wash and sample buffers, substrate buffer, stop solution and detailed protocols.
Specificity:
Bovine A2 beta-casein
Storage:
Store at 2-8°C
Range:
3.1 - 200 ng/mL
Sample Type:
Bovine Milk
Sensitivity:
This bovine A2 beta-casein ELISA kit detects a minimum of 2 ng/mL A2 beta-casein in assay buffer (defined as A1 concentration at blank OD plus 3x standard deviations of the blank OD (n=10)).
Cross Reactivity:
No cross-reactivity is observed with bovine A1 beta-Casein.
Store lyophilized/reconstituted at -20 °C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
Host Animal:
Rabbit
Species Reactivity:
Arabidopsis thaliana, Hordeum vulgare, Picea abies, Picea glauca, Pinus strobus, Spinacia oleracea, Drosera capensis. These antibodies have been shown to be reactive in all dicots, monocots, and gymnosperms
Immunogen:
BSA or KLH-conjugated synthetic peptides derived from conserved regions of plant Lhca1-4 and Lhcb1-6 protein sequences.
For detection of algal LHC proteins, we recommend: Set of 4 Chlamydomonas anti-Lhc antibodies
Application Details:
This set contains the following antibodies:Product number:Product name: AS01 005AS01 006AS01 007AS01 008AS01 004AS01 003AS01 002AS04 045AS01 009AS01 010Anti-Lhca1Anti-Lhca2Anti-Lhca3Anti-Lhca4Anti-LhcbAnti-Lhcb2Anti-Lhcb3Anti-Lhcb4Anti-Lhcb5Anti-Lhcb6Product AS01 009 and AS01 010 can be sold containing ProClin if requested.
Purity:
Total IgG. Protein G purified in PBS pH 7.4.: AS01 005, AS01 006, AS01 007, AS01 008, AS01 002, AS04 045 affinity purified serum: AS01 004, AS01 003. Serum: AS01 009, AS01 010.
Reconstitution:
For reconstitution add sterile water according to label on each tube
Molecular Weight:
20 - 29 kDa
Not reactive in:
No confirmed exceptions from predicted reactivity are currently known
Selected references:
Merry et al. (2017). A comparison of pine and spruce in recovery from winter stress; changes in recovery kinetics, and the abundance and phosphorylation status of photosynthetic proteins during winter. Tree Physiol. 2017 Sep 1;37(9):1239-1250. doi: 10.1093/treephys/tpx065.Li et al. (2017). NYEs/SGRs-mediated chlorophyll degradation is critical for detoxification during seed maturation in Arabidopsis. Plant J. 2017 Nov;92(4):650-661. doi: 10.1111/tpj.13710. Epub 2017 Oct 20.Yoshida et al. (2016). Hisabori T1.Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability. Proc Natl Acad Sci U S A. 2016 Jul 5;113(27):E3967-76. doi: 10.1073/pnas.1604101113. Epub 2016 Jun 22.Pavlovič et al. (2016). A carnivorous sundew plant prefers protein over chitin as a source of nitrogen from its traps. Plant Physiol Biochem. 2016 Mar 5;104:11-16. doi: 10.1016/j.plaphy.2016.03.008Xu et al. (2011). Light-harvesting chlorophyll a/b-binding proteins are required for stomatal response to abscisic acid in Arabidopsis. J. Ex. Bot. Dec 5 (ahead of print).Kang et al. (2010). Evaluation of light-harvesting complex proteins as senescence-related protein markers in detached rice leaves. Photosynthetica 47, 4:638-640.
10 mM Sodium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Preservative:
0.05% (w/v) Sodium Azide
Storage:
-20C or colder
Country Of Origin:
US Origin
Disclaimer:
For in vitro Laboratory Use Only. Not for diagnostic or therapeutic use. Not for human or animal consumption. Suggested applications of our products are not recommendations to use our products in violation of any patent or as a license under any patent of ImmunoReagents, Inc. Product may not be resold or modified for resale without prior written approval of ImmunoReagents, Inc.
Serine/Threonine-Protein Kinase B-Raf (BRAF) is a cytoplasmic serine-threonine kinase of the RAF family, which mediates downstream cellular responses to growth signals through the mitogen-activated protein kinase (MAPK) signaling pathway. Oncogenic mutations in the BRAF gene, 80% of which are a single V600E substitution within the kinase domain, constitutively activate the MAPK signaling pathway and result in increased cell proliferation and apoptosis resistance. The V600E mutation is observed in colorectal cancer, non-Hodgkin's lymphoma, papillary thyroid carcinoma, malignant melanoma, non-small-cell lung carcinoma, and lung adenocarcinoma. BRAF V600E is therefore an important immunohistochemical marker for tumour diagnosis and prognosis.
The Plus HRP Kits, Mouse is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus HRP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed via a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP123) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP124) forms a dark brown precipitate.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 500 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP123 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP124 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus HRP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRPconjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP132) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP133) forms a dark brown precipitate.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 500 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC Single Solution, AEC substrate kit, DAB Substrate kit, DAB High Contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Procedure:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP132 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP133 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 500 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 500 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
?-Casein is expressed as 13 genetic variants resulting from single nucleotide polymorphisms (SNP) in the CSN2 gene. The most frequent genetic variants in western dairy breeds are ?-Casein A1 and ?-Casein A2. The two types of ?-Casein protein, A1 and A2, differ by a single-point mutation at amino acid position 82 (P82/H82). The Biosensis Bovine A1 ?-Casein enzyme-linked immunosorbent assay (ELISA) Kit is a sandwich ELISA that allows the quantification of the bovine A1 ?-Casein isoform in 5 hours. This kit consists of a pre-coated rabbit anti-Bovine ?-Casein polyclonal capture antibody, a chicken anti-bovine A1 ?-Casein detection antibody and a horseradish peroxidase (HRP)-conjugated donkey anti-chicken IgY antibody. The addition of a substrate (3,3',5,5' -tetramethylbenzidine, TMB) yields a coloured reaction product which is directly proportional to the concentration of Bovine A1 ?-Casein present in samples and protein standards. Extensive validation has shown accurate quantification of A1 ?-Casein in UHT (Ultra High Temperature) treated milk or long life milk, organic pasteurized and homogenized full cream milk, fresh pasteurized and homogenized milk, cold-pressed raw milk (non-pasteurized, non-homogenized), and biodynamic full-cream whole milk. Please refer to the kit protocol for specific use instructions. The A2 isoform is not detected in this ELISA assay.
Product Type:
ELISA Assay
Species Reactivity:
Bovine
Immunogen:
Purified bovine beta Casein and peptide specific peptides for A1
Applications:
ELISA
Application Details:
ELISA. For the quantification of A1 Beta casein (A1) in Bovine Milk. Please download the detailed product insert for complete instructions for the successful use of this ELISA. Use only as directed.
Alternative Names:
CSN2; Bovine A1 Beta Casein; A1;
Biosensis Brand:
Biosensis®
Detection Method:
Colorimetric
Shelf Life:
12 months after date of receipt unopened.
Use:
For research use only.
Kit Components:
The ELISA kit box contains 96-well pre-coated strip plate(s), protein standards, detection reagents, wash and sample buffers, substrate buffer, stop solution and detailed protocols.
Specificity:
Bovine A1 beta-casein
Storage:
Store at 2-8°C
Range:
3.1 - 200 ng/mL
Sample Type:
Bovine Milk
Sensitivity:
This bovine A1 beta-casein ELISA kit detects a minimum of 3 ng/mL A1 beta-casein in assay buffer (defined as A1 concentration at blank OD plus 3x standard deviations of the blank OD (n=10))
Cross Reactivity:
No cross-reactivity is observed with bovine A2 beta-Casein
?-Casein is expressed as 13 genetic variants resulting from single nucleotide polymorphisms (SNP) in the CSN2 gene. The most frequent genetic variants in western dairy breeds are ?-Casein A1 and ?-Casein A2. The two types of ?-Casein protein, A1 and A2, differ by a single-point mutation at amino acid position 82 (P82/H82). The Biosensis Bovine A2 ?-Casein enzyme-linked immunosorbent assay (ELISA) Kit is a sandwich ELISA that allows the quantification of the bovine A2 ?-Casein isoform in 5 hours. This kit consists of a pre-coated rabbit anti-bovine ?-Casein polyclonal capture antibody, a chicken anti-bovine A2 ?-Casein detection antibody and a horseradish peroxidase (HRP)-conjugated donkey anti-chicken IgY antibody. The addition of a substrate (3,3',5,5' -tetramethylbenzidine, TMB) yields a coloured reaction product which is directly proportional to the concentration of Bovine A2 ?-Casein present in samples and protein standards. Extensive validation has shown accurate quantification of A2 ?-Casein in full cream milk, skim milk and reconstituted A2 milk powder. Please refer to the kit protocol for specific use instructions. The A1 isoform is not detected in this ELISA assay.
Product Type:
ELISA Assay
Species Reactivity:
Bovine
Immunogen:
Purified bovine beta Casein and peptide specific peptides for A2
Applications:
ELISA
Application Details:
ELISA. For the quantification of A2 Beta casein (A2) in Bovine Milk. Please download the detailed product insert for complete instructions for the successful use of this ELISA. Use only as directed.
Alternative Names:
CSN2; Bovine A2 Beta Casein; A2;
Biosensis Brand:
Biosensis®
Detection Method:
Colorimetric
Shelf Life:
12 months after date of receipt unopened.
Use:
For research use only.
Kit Components:
The ELISA kit box contains 96-well pre-coated strip plate(s), protein standards, detection reagents, wash and sample buffers, substrate buffer, stop solution and detailed protocols.
Specificity:
Bovine A2 beta-casein
Storage:
Store at 2-8°C
Range:
3.1 - 200 ng/mL
Sample Type:
Bovine Milk
Sensitivity:
This bovine A2 beta-casein ELISA kit detects a minimum of 2 ng/mL A2 beta-casein in assay buffer (defined as A1 concentration at blank OD plus 3x standard deviations of the blank OD (n=10))
Cross Reactivity:
No cross-reactivity is observed with bovine A1 beta-Casein
For use as a standard or control in most immunoassay formats
Purity:
Immunogen affinity purified donkey IgG.
Special application note:
Antibody purity is > 95% based on SDS-PAGE.Affinity purified using solid phase Mouse IgG .Antibody is supplied in 10 mM Sodium Phosphate, 0.15 M Sodium Chloride, pH 7.2. Based on IEP, this antibody reacts with: heavy (γ) chains on mouse IgG light chains on all mouse immunoglobulins . Based on IEP, no reactivity is observed to: non-immunoglobulin mouse serum immunoglobulins .
The Plus AP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus AP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kits, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Broad Spectrum is polyvalent. With this kit it is therefore possible to detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized via incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP110) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
500 ml Blocking Solution Reagent 1 (ready-to-use) 500 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 500 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion.Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP110 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (substrate buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 12. Wash with distilled H2O 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolized by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see also Limitations of the procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3), used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
5-methylcytosine (5-mC) is formed from the DNA methylation of the 5-carbon found on the cytosine ring. A 5-methylcytosine monoclonal antibody is a useful tool in identifying and discriminating between the unmodified cytosine base (C) and the methylated cytosine base (5-mC) as part of DNA methylation studies. DNA methylation plays an important role in the repression of transcription in the genome. When present in promoter regions, 5-mC is associated with stable transcriptional silencing which results in inactivation of gene function, thereby having an important role in tumorigenesis.
5-methylcytosine (5-mC) is formed from the DNA methylation of the 5-carbon found on the cytosine ring. A 5-methylcytosine monoclonal antibody is a useful tool in identifying and discriminating between the unmodified cytosine base (C) and the methylated cytosine base (5-mC) as part of DNA methylation studies. DNA methylation plays an important role in the repression of transcription in the genome. When present in promoter regions, 5-mC is associated with stable transcriptional silencing which results in inactivation of gene function, thereby having an important role in tumorigenesis.
Tris Buffer pH7.6 with BSA, and sodium azide as preservative
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