The Plus HRP Polymer anti-Rabbit is designed for the qualitative detection of antigens in fixed paraffinembedded 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 rabbits. 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-Rabbit 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 horse radish peroxidase (HRP). 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 rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP Polymer anti-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. 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 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 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 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 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-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 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 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 (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-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 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 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. 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). 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. 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 reagent. 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 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
The blocking solution Peroxide Block is intended for inhibition of endogenous peroxidase activity in tissue sections. It is primarily intended to be used in immunohistochemistry on formalin-fixed paraffin-embedded samples if using a detection system with horse radish peroxidase.
Endogenous peroxidase activity in the tissue can result in unspecific background staining in immunohistochemical staining procedures using horse radish peroxidase (HRP) as detection enzyme. This effect can be eliminated when tissue sections are incubated with Peroxide Block prior to immunohistochemical staining. Hydrogen peroxide in the solution blocks the activity of endogenous peroxidase.
Principle of method:
Peroxide Block is applied onto tissue sections to reduce non-specific staining due to endogenous peroxidase activity in immunohistochemistry. The step is carried out before incubation with primary antibody but after dewaxing and rehydration. If a heat induced epitope retrieval (HIER) or enzymatic digestion is necessary for immunohistochemical detection it is of no importance if the Peroxide Block is used before or after this step. In some cases it has been shown, that blocking of endogenous peroxidase before the epitope retrieval leads to better results.
Reagents provided:
500 ml Peroxide Block (ready-to-use)
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. Avoid exposure to strong light. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product 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 this reagent, please contact our technical support.
Procedure:
1. Apply Peroxide Block for 10 minutes at room temperature. The section should be covered completely. 2. Rinse with wash buffer. 3. Proceed with next steps for immunohistochemical staining as usual starting with protein blocking or the primary antibody.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 please read these instructions carefully, contact our technical support. Also refer to the instructions of the detection systems containing Peroxide Block 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 binding. 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen 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 may occur. A material safety data sheet (MSDS) is available upon request
The BrightDiluents universal IHC diluent is specially formulated to stabilize antibodies and eliminate backgrounds encountered in immunohistochemistry staining procedures. The diluent contains complex proteins and non-protein mixtures which eliminate histostaining background by blocking Fc receptors and prevents non-specific protein-protein interactions. It can also be used as blocking applied before primary antibodies. If primary antibodies are diluted in this diluent, a blocking step before primary antibodies can generally be omitted. The blocking /diluent solution is supplied in Ready-to-Use format. It should not be diluted to be effective. The diluent contains no azide, therefore, it can be used to dilute and stabilize HRP-conjugates as well as any other solution that need and stabilizing carrier protein, such as Blocking and Poly-AP conjugates. This product should be interpreted by a qualified pathologist with relevant clinical information, morphological and histological studies and with proper controls. The ready-to-use antibody diluent is available in different colors: MON-APP917 (Transparant/colorless), MON-APP917B (Blue), MON-APP917Y (Yellow), MON-APP917G (green), MON-APP917R (Red)
Principle of method:
Antibody diluent
Reagents provided:
125 ml BrightDiluent, normal antibody diluent (ready-to-use)
The BrightDiluents universal IHC diluent is specially formulated to stabilize antibodies and eliminate backgrounds encountered in immunohistochemistry staining procedures. The diluent contains complex proteins and non-protein mixtures which eliminate histostaining background by blocking Fc receptors and prevents non-specific protein-protein interactions. It can also be used as blocking applied before primary antibodies. If primary antibodies are diluted in this diluent, a blocking step before primary antibodies can generally be omitted. The blocking /diluent solution is supplied in Ready-to-Use format. It should not be diluted to be effective. The diluent contains no azide, therefore, it can be used to dilute and stabilize HRP-conjugates as well as any other solution that need and stabilizing carrier protein, such as Blocking and Poly-AP conjugates. This product should be interpreted by a qualified pathologist with relevant clinical information, morphological and histological studies and with proper controls. The ready-to-use antibody diluent is available in different colors: MON-APP917 (Transparant/colorless), MON-APP917B (Blue), MON-APP917Y (Yellow), MON-APP917G (green), MON-APP917R (Red)
Principle of method:
Antibody diluent
Reagents provided:
125 ml BrightDiluent, normal antibody diluent (ready-to-use)
The Citrate buffer, is designed for use during heat-induced epitope retrieval (HIER) on formalinfixed paraffin-embedded tissue sections prior to antibody application. In immunohistochemistry (IHC), protein cross-links are formed during formalin or other aldehyde fixation. These cross-links mask the antigenic sites in tissue specimens and results in weak or false negative staining in immunohistochemical detection of proteins. The use of this citrate buffer in combination with heat ensures that the antigenicity of proteins modified, thus enhancing staining intensity of antibodies. For the recommended pre-treatment technique, please refer to the individual antibody's instructions for use. This buffer is a 10x concentrate solution and must be diluted 1:10 with deionized water before using. The final readyto-use solution should have a pH of 6.0 ± 0.3. The buffer can be used for a maximum of three runs within five days after dilution. Also available in 250 ml, 500 ml en 1000 ml.
Principle of method:
Heat-Induced Epitope Retrieval
Reagents provided:
100 ml 10x concentrate solution. Citrate buffer, Heat-Induced Epitope Retrieval (10x, Tween20), Red
Storage and handling:
Store at room temperature
Reagent preparation:
Before use dilute 1:10 with deionized water. The final ready-to-use solution should have a pH of 6.0 ± 0,3,
Procedure:
1. Deparaffinize and rehydrate the tissue slides. 2. Fill the PT Module tank with 150 ml of citrate buffer and 1,350 ml of deionized water, to obtain 1,500 ml of ready-to-use solution (1:10 dilution). 3. Pre-heat the PT Module until temperature reaches 65°C and place the formalin-fixed slides in slide racks into the PT Module tank. 4. Heat the citrate buffer and slides to 98°C and incubate for 20 minutes. 5. Cool slides in the PT Module to 65°C. 6. Remove slides and cool to room temperature for at least 5 minutes in a PBS or TBS based buffer. 7. Continue with staining according to IHC protocol. Note: Alternative heating sources, such as a microwave or a pressure cooker, can be used to replace the PT Module tank. The optimal incubation time for these heating sources should be determined by authorised personnel / researchers.
Antibody Diluent B is especially developed for dilution of certain primary antibodies. Antibodies diluted with Antibody Diluent B are primarily used in immunohistochemistry with formalin-fixed paraffin-embedded tissue sections, but also with frozen, HOPE-fixed, and cytological samples as well as in immunoblot procedures.
Antibody diluents used in immunohistochemistry should protect the antibody from microbial contamination and stabilize the antibody chemically. Antibody Diluent B reduces non-specific binding of antibodies to tissue sections and is therefore extremely useful in receiving background-free staining results.
Principle of method:
Immunohistochemical staining procedures often start with incubation of a blocking solution to reduce unspecific binding of primary antibody to tissue sections. This step can be omitted if the antibody used is diluted in Antibody Diluent B. Antibody Diluent B minimises unspecific binding of the primary antibody to the tissue section, reduces surface tension of the antibody solution and improves spreading the reagent on the slide, increases microbial and chemical stability of the antibody, reduces adhesion of antibody to the surface of the vial, and minimises the danger of antibody degradation by proteolytic enzymes.
Reagents provided:
25 ml Antibody Diluent B (ready-to-use)
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. Do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. If stored at room temperature the solution is stable for at least 10 month from the date of delivery. 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 this reagent, please contact our technical support.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 please read these instructions carefully, contact our technical support. Also refer to the instructions of the detection systems containing Peroxide Block 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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.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 reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen 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 may occur. (NaN3), used for stabilisation, is not considered hazardous material in the concentration used. 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. A material safety data sheet (MSDS) is available upon request.
The BrightDiluents universal IHC diluent is specially formulated to stabilize antibodies and eliminate backgrounds encountered in immunohistochemistry staining procedures. The diluent contains complex proteins and non-protein mixtures which eliminate histostaining background by blocking Fc receptors and prevents non-specific protein-protein interactions. It can also be used as blocking applied before primary antibodies. If primary antibodies are diluted in this diluent, a blocking step before primary antibodies can generally be omitted. The blocking /diluent solution is supplied in Ready-to-Use format. It should not be diluted to be effective. The diluent contains no azide, therefore, it can be used to dilute and stabilize HRP-conjugates as well as any other solution that need and stabilizing carrier protein, such as Blocking and Poly-AP conjugates. This product should be interpreted by a qualified pathologist with relevant clinical information, morphological and histological studies and with proper controls. The ready-to-use antibody diluent is available in different colors: MON-APP917 (Transparant/colorless), MON-APP917B (Blue), MON-APP917Y (Yellow), MON-APP917G (green), MON-APP917R (Red)
Principle of method:
Antibody diluent
Reagents provided:
125 ml BrightDiluent, normal antibody diluent (ready-to-use)
The Citrate buffer, is designed for use during heat-induced epitope retrieval (HIER) on formalinfixed paraffin-embedded tissue sections prior to antibody application. In immunohistochemistry (IHC), protein cross-links are formed during formalin or other aldehyde fixation. These cross-links mask the antigenic sites in tissue specimens and results in weak or false negative staining in immunohistochemical detection of proteins. The use of this citrate buffer in combination with heat ensures that the antigenicity of proteins modified, thus enhancing staining intensity of antibodies. For the recommended pre-treatment technique, please refer to the individual antibody's instructions for use. This buffer is a 10x concentrate solution and must be diluted 1:10 with deionized water before using. The final readyto-use solution should have a pH of 6.0 ± 0.3. The buffer can be used for a maximum of three runs within five days after dilution. Also available in 250 ml, 500 ml en 1000 ml.
Before use dilute 1:10 with deionized water. The final ready-to-use solution should have a pH of 6.0 ± 0,3,
Procedure:
1. Deparaffinize and rehydrate the tissue slides. 2. Fill the PT Module tank with 150 ml of citrate buffer and 1,350 ml of deionized water, to obtain 1,500 ml of ready-to-use solution (1:10 dilution). 3. Pre-heat the PT Module until temperature reaches 65°C and place the formalin-fixed slides in slide racks into the PT Module tank. 4. Heat the citrate buffer and slides to 98°C and incubate for 20 minutes. 5. Cool slides in the PT Module to 65°C. 6. Remove slides and cool to room temperature for at least 5 minutes in a PBS or TBS based buffer. 7. Continue with staining according to IHC protocol. Note: Alternative heating sources, such as a microwave or a pressure cooker, can be used to replace the PT Module tank. The optimal incubation time for these heating sources should be determined by authorised personnel / researchers.
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:
6 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.
The TRIS/EDTA buffer, is designed for use during heat-induced epitope retrieval (HIER) on formalinfixed paraffin-embedded tissue sections prior to antibody application. In immunohistochemistry (IHC), protein cross-links are formed during formalin or other aldehyde fixation. These cross-links mask the antigenic sites in tissue specimens and results in weak or false negative staining in immunohistochemical detection of proteins. The use of this citrate buffer in combination with heat ensures that the antigenicity of proteins modified, thus enhancing staining intensity of antibodies. For the recommended pre-treatment technique, please refer to the individual antibody's instructions for use. This buffer is a 10x concentrate solution and must be diluted 1:10 with deionized water before using. The final readyto-use solution should have a pH of 9.0 ± 0.3. The buffer can be used for a maximum of three runs within five days after dilution. Also available in 250 ml, 500 ml en 1000 ml.
Principle of method:
Heat-Induced Epitope Retrieval
Reagents provided:
100 ml 10x concentrate solution. TRIS/EDTA buffer, Heat-Induced Epitope Retrieval (10x, Tween20), Blue
Storage and handling:
Store at room temperature
Reagent preparation:
Before use dilute 1:10 with deionized water. The final ready-to-use solution should have a pH of 9.0 ± 0,3,
Procedure:
1. Deparaffinize and rehydrate the tissue slides. 2. Fill the PT Module tank with 150 ml of TRIS?EDTA buffer and 1,350 ml of deionized water, to obtain 1,500 ml of ready-to-use solution (1:10 dilution). 3. Pre-heat the PT Module until temperature reaches 65°C and place the formalin-fixed slides in slide racks into the PT Module tank. 4. Heat the TRIS/EDTA buffer and slides to 98°C and incubate for 15 minutes. 5. Cool slides in the PT Module to 65°C. 6. Remove slides and cool to room temperature for at least 5 minutes in a PBS or TBS based buffer. 7. Continue with staining according to IHC protocol. Note: Alternative heating sources, such as a microwave or a pressure cooker, can be used to replace the PT Module tank. The optimal incubation time for these heating sources should be determined by authorised personnel / researchers.
The TRIS/EDTA buffer, is designed for use during heat-induced epitope retrieval (HIER) on formalinfixed paraffin-embedded tissue sections prior to antibody application. In immunohistochemistry (IHC), protein cross-links are formed during formalin or other aldehyde fixation. These cross-links mask the antigenic sites in tissue specimens and results in weak or false negative staining in immunohistochemical detection of proteins. The use of this citrate buffer in combination with heat ensures that the antigenicity of proteins modified, thus enhancing staining intensity of antibodies. For the recommended pre-treatment technique, please refer to the individual antibody's instructions for use. This buffer is a 10x concentrate solution and must be diluted 1:10 with deionized water before using. The final readyto-use solution should have a pH of 9.0 ± 0.3. The buffer can be used for a maximum of three runs within five days after dilution. Also available in 250 ml, 500 ml en 1000 ml.
Before use dilute 1:10 with deionized water. The final ready-to-use solution should have a pH of 9.0 ± 0,3,
Procedure:
1. Deparaffinize and rehydrate the tissue slides. 2. Fill the PT Module tank with 150 ml of TRIS?EDTA buffer and 1,350 ml of deionized water, to obtain 1,500 ml of ready-to-use solution (1:10 dilution). 3. Pre-heat the PT Module until temperature reaches 65°C and place the formalin-fixed slides in slide racks into the PT Module tank. 4. Heat the TRIS/EDTA buffer and slides to 98°C and incubate for 15 minutes. 5. Cool slides in the PT Module to 65°C. 6. Remove slides and cool to room temperature for at least 5 minutes in a PBS or TBS based buffer. 7. Continue with staining according to IHC protocol. Note: Alternative heating sources, such as a microwave or a pressure cooker, can be used to replace the PT Module tank. The optimal incubation time for these heating sources should be determined by authorised personnel / researchers.
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.
Antibody Diluent B is especially developed for dilution of certain primary antibodies. Antibodies diluted with Antibody Diluent B are primarily used in immunohistochemistry with formalin-fixed paraffin-embedded tissue sections, but also with frozen, HOPE-fixed, and cytological samples as well as in immunoblot procedures.
Antibody diluents used in immunohistochemistry should protect the antibody from microbial contamination and stabilize the antibody chemically. Antibody Diluent B reduces non-specific binding of antibodies to tissue sections and is therefore extremely useful in receiving background-free staining results.
Principle of method:
Immunohistochemical staining procedures often start with incubation of a blocking solution to reduce unspecific binding of primary antibody to tissue sections. This step can be omitted if the antibody used is diluted in Antibody Diluent B. Antibody Diluent B minimises unspecific binding of the primary antibody to the tissue section, reduces surface tension of the antibody solution and improves spreading the reagent on the slide, increases microbial and chemical stability of the antibody, reduces adhesion of antibody to the surface of the vial, and minimises the danger of antibody degradation by proteolytic enzymes.
Reagents provided:
100 ml Antibody Diluent B (ready-to-use)
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. Do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. If stored at room temperature the solution is stable for at least 10 month from the date of delivery. 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 this reagent, please contact our technical support.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 please read these instructions carefully, contact our technical support. Also refer to the instructions of the detection systems containing Peroxide Block 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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.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 reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen 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 may occur. (NaN3), used for stabilisation, is not considered hazardous material in the concentration used. 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. A material safety data sheet (MSDS) is available upon request.
Storage of tissue samples. The aluminium cryo vials with screw caps can be used for storage of tissue samples. The cryo tubes are suitable for storage of tissue samples in liquid nitrogen. Suitable for deepfreezing till -196°C. Autoclavable at 121°C . Available in 3ml (PA6003) and 15ml (PA6015) (sets of 100 pieces).
Storage of cryo tubes in liquid nitrogen: 1) Place the tissue in the cryo tube, 2) Close the cryotube tighly and place the cryotube in the liquid nitrogen for 1 minute, 3) The cryo vials can be stored at -80°C. Use of cryo tube in combination with isopentane: 1)Place a cup with isopentane in the liquid nitrogen; after approximately 2 minutes the isopentane clot and have a temperature of -160°C. 2)Place the tissue sample or biopsy in the isopentane for 30 seconds, 3) Place the tissue in the cryo vials and store at -80°C.
Reagent preparation:
Make sure that the cap and the vial have the same temperature because the material could slightly shrink or expand under influence of excessive temperature differences.
Expected results:
Long term storage of tissue
Precautions:
Wear a face mask at all times, Check if the screw treads and the lid are okay, Screw the lid on tightly. Attention: AVOID EXCESSIVE TEMPERATURE CHANGES, the cap could explode from the cryo tube.
Storage of tissue samples. The aluminium cryo vials with screw caps can be used for storage of tissue samples. The cryo tubes are suitable for storage of tissue samples in liquid nitrogen. Suitable for deepfreezing till -196°C. Autoclavable at 121°C . Available in 3ml (PA6003) and 15ml (PA6015) (sets of 100 pieces).
Storage of cryo tubes in liquid nitrogen: 1) Place the tissue in the cryo tube, 2) Close the cryotube tighly and place the cryotube in the liquid nitrogen for 1 minute, 3) The cryo vials can be stored at -80°C. Use of cryo tube in combination with isopentane: 1)Place a cup with isopentane in the liquid nitrogen; after approximately 2 minutes the isopentane clot and have a temperature of -160°C. 2)Place the tissue sample or biopsy in the isopentane for 30 seconds, 3) Place the tissue in the cryo vials and store at -80°C.
Reagent preparation:
Make sure that the cap and the vial have the same temperature because the material could slightly shrink or expand under influence of excessive temperature differences.
Expected results:
Long term storage of tissue
Precautions:
Wear a face mask at all times, Check if the screw treads and the lid are okay, Screw the lid on tightly. Attention: AVOID EXCESSIVE TEMPERATURE CHANGES, the cap could explode from the cryo tube.
Pepsin Solution is a ready-to-use solution developed for enzymatic epitope retrieval on formalin-fixed tissue sections on slides. This procedure (sometimes called PIER, Protease Induced Epitope Retrieval) is primarily used in immunohistochemical staining procedures.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to antigens the epitopes have to be recovered. Enzymatic digestion with proteolytic enzymes (PIER) restores structures of the epitopes making them more accessible to specific antibodies. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
Pepsin Solution is a ready-to-use stabilised pepsin solution for enzymatic epitope retrieval.
Reagents provided:
60 ml Pepsin Solution (Ready-To-Use)
Storage and handling:
The solution should be stored at 2-8°C without furt her dilution. Do not freeze it. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product 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 this reagent, please contact our technical support
Reagent preparation:
The solution is ready-to-use.
Procedure:
Pepsin Solution is suitable for enzymatic epitope retrieval carried out after the dewaxing and rehydration of the tissue sections. 1. Cover deparaffinised and rehydrated tissue sections with ready-to-use Pepsin Solution. 2. Incubate for 10 - 15 minutes at 37°C or 20 30 minutes at room temperature. The optimal incubation time needs to be elaborated by the operator. It was shown that in individual cases a stronger signal can be obtained when the incubation time is elongated up to 120 minutes (e. g. for detection of Collagen IV with different antibodies). 3. Rinse carefully (3 x) with wash buffer. 4. Proceed with immunohistological staining as usual.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 please read these instructions carefully, 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 reagent and specimen with eye, skin or mucous membranes. In case of reagent or specimen coming into contact with a sensitive area, wash area with large amounts of water. A material safety data sheet (MSDS) is available upon request.
Pronase Solution is a ready-to-use solution developed for enzymatic epitope retrieval on formalin-fixed tissue sections on slides. This procedure (sometimes called PIER, Protease Induced Epitope Retrieval) is primarily used in immunohistochemical staining procedures. Proteolytic pre-treatments are also used in protocols for in situhybridization. Pronase solution is intended for research use only.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to antigens the epitopes have to be recovered. Enzymatic digestion with proteolytic enzymes (PIER) restores structures of the epitopes making them more accessible to specific antibodies. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
Pronase Solution is a ready-to-use solution for enzymatic epitope retrieval.
Reagents provided:
250 ml Pronase Solution (Ready-To-Use)
Storage and handling:
The solution should be stored in aliquots at -20°C without further dilution. The solution should be aliquoted in order to avoid repeated freeze and thawing. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date.
Reagent preparation:
Pronase solution is ready-to-use and should be at room temperature prior to use.
Procedure:
Pronase Solution is suitable for enzymatic epitope retrieval carried out after the dewaxing and rehydration of the tissue sections. 1. Cover deparaffinised and rehydrated tissue sections with ready-to-use Pronase Solution. 2. Incubate for 15 - 20 minutes at room temperature. The optimal incubation time needs to be elaborated by the operator. 3. Rinse carefully (3 x), first with distilled water followed by buffer. 4. Proceed with immunohistological staining as usual.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 please read these instructions carefully, contact our technical support
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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 the reagent with eye, skin or mucous membrane. In case of reagent coming into contact with a sensitive area, wash the area with large amounts of water. Material safety data sheets (MSDS) are available upon request.
Trypsin Pretreatment Kit consists of 2 reagents for the preparation of a trypsin solution used for enzymatic epitope retrieval on formalin-fixed tissue sections on slides. This procedure (sometimes called PIER, Protease Induced Epitope Retrieval) is primarily used in immunohistochemical staining procedures. Trypsin Pretreatment Kit is for research use only.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to antigens the epitopes have to be recovered. Enzymatic digestion with proteolytic enzymes (PIER) restores structures of the epitopes making them more accessible to specific antibodies. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
The components of this Trypsin Pretreatment Kit allow for the preparation of a buffered trypsin solution for enzymatic epitope retrieval.
Reagents provided:
30 ml Trypsin Solution 125 ml Trypsin Buffer
Storage and handling:
The solutions should be stored at 2-8°C. Please sto re 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. 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 this reagent, please contact our technical support.
Reagent preparation:
Mix 1 part Trypsin Solution with 3 parts Trypsin Buffer. The activity of the resulting trypsin solution can be adjusted by variation of the mixing ratio. Mix the two components in the ratio 1:1 when strong epitope retrieval is desired. The working solution is stable for at least one week if stored at 2-8°C.
Procedure:
Trypsin Pretreatment Kit is suitable for enzymatic epitope retrieval carried out after the dewaxing and rehydration of the sections. 1. Cover deparaffinised and rehydrated tissue sections with trypsin working solution. 2. Incubate for 10 - 20 minutes at 37°C. 3. Rinse carefully (3 x) with wash buffer. 4. Proceed with immunohistological staining as usual.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 reagent and specimen with eye, skin or mucous membranes. In case of reagent or specimen coming into contact with a sensitive area, wash area with large amounts of water. A material safety data sheet (MSDS) is available upon request.
Fast Enzyme is a ready-to-use solution developed for enzymatic epitope retrieval on formalin-fixed tissue sections on slides. This procedure (sometimes called PIER, Protease Induced Epitope Retrieval) is primarily used in immunohistochemical staining procedures. Proteolytic pre-treatment is also used in in situ-hybridisation. Fast Enzyme is intended for research use only.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to antigens the epitopes have to be recovered. Enzymatic digestion with proteolytic enzymes (PIER) restores structures of the epitopes making them more accessible to specific antibodies. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
Fast Enzyme is a ready-to-use enzyme solution for enzymatic epitope retrieval.
Reagents provided:
15 ml Fast Enzyme (Ready-To-Use)
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product 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 this reagent, please contact our technical support.
Reagent preparation:
The solution is ready-to-use.
Procedure:
Fast Enzyme is suitable for enzymatic epitope retrieval carried out after the dewaxing and rehydration of the tissue sections. 1. Cover deparaffinised and rehydrated tissue sections with ready-to-use Fast Enzyme Solution. 2. Incubate for 5 minutes at room temperature. (It was shown that in individual cases a stronger signal can be obtained when the incubation time is elongated. Usually an incubation for 5 min at room temperatur is sufficient.) 3. Rinse carefully (3 x) with wash buffer. 4. Proceed with immunohistological staining as usual.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 reagent and specimen with eye, skin or mucous membranes. In case of reagent or specimen coming into contact with a sensitive area, wash area with large amounts of water. A material safety data sheet (MSDS) is available upon request.
HIER Citrate Buffer pH 6.0 is a solution developed for heat induced epitope retrieval (HIER) in formalin-fixed paraffin-embedded tissue sections on slides. This procedure is primarily used in immunohistochemistry.
Immunohistochemical staining procedures consist of sequential incubation steps with blocking solutions, antibodies and secondary reagents, enzymes and chromogenic substrates carried out on tissue sections. These tissue sections are mostly prepared out of formalin-fixed paraffin-embedded tissue blocks. Cellular structures are very effectively stabilised by formalin fixation which results in optimal morphological preservation of the sample. On the other hand the formalin fixation leads to strong cross-links between proteins. This means that epitopes of antigens are being masked and often are no longer accessible for primary antibodies. In order to enable primary antibodies to bind to the antigens the epitopes have to be recovered. Heat induced epitope retrieval (HIER) in buffer solutions of different compositions and pH-values restore structures of the epitopes making them more accessible to specific antibodies. Enzymatic digestion with proteolytic enzymes is another way of recovering epitopes. The primary antibody used determines the appropriate method.
Principle of method:
HIER Citrate Buffer pH 6.0 is a 10fold concentrated citrate buffer solution with additives of stabilising substances. For preparation of the working strength solution the buffer concentrate is diluted 1:10 with deionised or distilled water. The resulting solution has a pH of 6.0 (5.8 to 6.2). HIER Citrate Buffer pH 6.0 is a very efficient epitope retrieval solution in immunohistochemical staining procedures to be used with primary antibodies of many different specificities.
Reagents provided:
100 ml HIER Citrate Buffer pH 6.0 (10fold concentrated, adequate for 1 litre ready-to-use citrate buffer)
Storage and handling:
The solution should be stored at 2-8°C. Do not free ze it. Under these conditions the solution is stable up to the expiry date indicated on the label. Do not use product after the expiry date. If stored at room temperature the solution is stable for at least 10 month from the date of delivery. The prepared working strength solution is stable for 1 month, if stored at 2-8°C. A positive and a negat ive 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 this reagent, please contact our technical support.
Reagent preparation:
Preparation of the citrate buffer working strength solution: Dilute HIER Citrate Buffer concentrate 1:10 with deionised or distilled water and mix thoroughly. The pH-value should be at 6.0 (5.8 to 6.2). If necessary adjust pH-value with diluted NaOH or HCl solution.
Procedure:
HIER Citrate Buffer is suitable for various HIER-methods such as steamer, pressure cooker, autoclave, water bath, and microwave oven. Tissue sections used in heat induced epitope retrieval should always be placed on adhesive slides. Epitope retrieval is carried out after dewaxing and rehydration of the sections. Exemplary protocol using steamer: 1. Prepare the working strength solution by diluting the buffer concentrate as described above and transfer to a Coplin jar. Please make sure that there is enough volume to cover the tissue sections on the slides completely. 2. Fill steamer with water according to instruction manual, close lid and start. 3. After 10 minutes place Coplin jar with citrate buffer in the steamer, close the lid and heat the solution for 20 minutes. 4. Place slides with tissue sections into the preheated solution and close the lid. Tissue sections have to be completely covered with citrate buffer solution. 5. Incubate slides 20 40 minutes. The optimal incubation time needs to be elaborated by the operator. 6. After the incubation take the Coplin jar with slides out of steamer and let cool down at room temperature for about 20 minutes. 7. Remove citrate buffer, rinse slides with wash buffer and proceed with immunohistological staining.
Expected results:
During the reaction of the substrate with horse radish peroxidase or 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 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 binding. 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 technique involving both histological and immunological detection methods. It requires a highly trained histotechnologist. 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. 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 reagent or specimen with eye, skin or mucous membrane. In case of the reagent or specimen 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 may occur. ProClin 300 is used for stabilisation. A material safety data sheet (MSDS) is available upon request.
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:
6 ml Blocking Solution Reagent 1 (ready-to-use) 6 ml PostBlock Reagent 2 (ready-to-use) 6 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.
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.
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:
6 ml Blocking Solution Reagent 1 (ready-to-use) 6 ml PostBlock Reagent 2 (ready-to-use) 6 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 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 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:
6 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.
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