The Plus HRP One-Step Polymer anti-Mouse/Rabbit is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. It was developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. The kit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP One-Step Polymer anti-Mouse/Rabbit is a highly sensitive detection reagent intended for use in immunohistochemistry and immunocytochemistry. The enzyme polymer consists of several molecules of secondary antibodies covalently bound to several molecules of horseradish peroxidase (HRP). Visualisation occurs via an enzyme-substrate reaction in the presence of a colorising reagent which permits microscopical analysis. The test system is suitable for the detection of mono- and polyclonal primary antibodies and sera obtained from mice or rabbit. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus HRP One-Step Polymer kits avoid the problem of background staining caused by endogenous biotin in the tissue.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (peroxide block). Background staining caused by unspecific binding of the primary antibody or the secondary antibody in the HRP One-Step Polymer is minimized by incubation with a protein blocking solution. This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the HRP One-Step Polymer is applied and incubated. Any excess of unbound polymer is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB forms a dark brown precipitate.
Reagents provided:
6 mL HRP One-Step-Polymer anti-Mouse/Rabbit (ready-to-use) Substrate systems recommended: Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB High contrast kit. Materials required but not supplied: Positive and negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out.
Procedure:
1. Peroxide blocking (3 % H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 5 min. 7. HRP One-Step Polymer anti-Mouse/Rabbit 30 min. 8. Washing with wash buffer 3 x 2 min. 9. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 10. Stopping the reaction with distilled H2O when the desired colour intensity is attained 11. Counterstaining and blueing 12. Mounting: aqueous with AEC, permanent with DAB
Expected results:
During the reaction of the substrate with horseradish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse or rabbit but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Nonspecific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme polymer or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use Blocking Solution or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase in the tissue. Maybe the hydrogen peroxide solution used for blocking was inactivated
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity may cause non-specific staining. The enzyme activity is blocked by incubation with hydrogen peroxide solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horseradish peroxidase) detection systems (Omata et al, 1980). Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution can result in decreasing signal intensity. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 950 is used for stabilisation. A Material safety data sheet (MSDS) is available upon request.
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:
6 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 Plus AP Polymer anti-Rabbit 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 monoand 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 AP 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 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 rabbits. In contrast to other detection techniques, which often use the streptavidin-biotin system the Plus AP 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. 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 Permanent AP Red leads to the formation of a magentared product of reaction at the place of the target antigen.
Reagents provided:
6 ml AP-Polymer anti-Rabbit (Ready-to-use) Substrate systems recommended: Permanent AP Red 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 solutions 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 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, 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 Rabbit 30 min. 6. Washing with wash buffer 3 x 2 min. 7. Permanent AP Red 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: 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 read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme 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 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 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.
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 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 HRP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus HRP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRPconjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP132) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP133) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml DAB Substrate Buffer 3 ml DAB Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC Single Solution, AEC substrate kit, DAB Substrate kit, DAB High Contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Procedure:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP132 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP133 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus HRP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRPconjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP132) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP133) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml AEC Substrate Buffer 3 ml AEC Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC Single Solution, AEC substrate kit, DAB Substrate kit, DAB High Contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Procedure:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP132 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP133 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 8 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) 8 x 5 ml Permanent Red Buffer (Substrate Buffer) 2 ml Permanent Red Concentrate (Chromogen) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus HRP Kits, Mouse is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus HRP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed via a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP123) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP124) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml DAB Substrate Buffer 3 ml DAB Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP123 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP124 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kits, Mouse is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus HRP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed via a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP123) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP124) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml AEC Substrate Buffer 3 ml AEC Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP123 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP124 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 8 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) 8 x 5 ml Permanent Red Buffer (Substrate Buffer) 2 ml Permanent Red Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
The Plus HRP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus HRP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Broad Spectrum is polyvalent. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP114) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP115) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml DAB Substrate Buffer 3 ml DAB Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP114 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP115 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus HRP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Broad Spectrum is polyvalent. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP114) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP115) forms a dark brown precipitate.
Reagents provided:
8 ml Peroxide Block (ready-to-use) 8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 8 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) 7 x 5 ml AEC Substrate Buffer 3 ml AEC Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support.
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP114 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP115 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus AP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus AP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kits, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Broad Spectrum is polyvalent. With this kit it is therefore possible to detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized via incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP110) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
8 ml Blocking Solution Reagent 1 (ready-to-use) 8 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 8 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) 8 x 5 ml Permanent Red Buffer (Substrate Buffer) 2 ml Permanent Red Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion.Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP110 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (substrate buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 12. Wash with distilled H2O 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolized by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see also Limitations of the procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3), used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus HRP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus HRP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRPconjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP132) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP133) forms a dark brown precipitate.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC Single Solution, AEC substrate kit, DAB Substrate kit, DAB High Contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Procedure:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP132 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP133 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
The Plus HRP Kits, Mouse is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus HRP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed via a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP123) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP124) forms a dark brown precipitate.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP123 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP124 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus AP Kit, Broad Spectrum is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig. The Plus AP Kit, Broad Spectrum can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kits, Broad Spectrum is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Broad Spectrum is polyvalent. With this kit it is therefore possible to detect mono- and polyclonal primary antibodies and sera obtained from mouse, rabbit, rat, and guinea pig.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized via incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP110) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
4x 15 ml Blocking Solution Reagent 1 (ready-to-use) 4x 15 ml Biotinylated Secondary Antibody, polyvalent Reagent 2 (ready-to-use) 4x 15 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) 8 x 5 ml Permanent Red Buffer (Substrate Buffer) 2 ml Permanent Red Concentrate (Chromogen) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solution should be stored at 2-8°C without further dilution. Please store the reagent in a dark place and do not freeze it. Under these conditions the solution is stable up to the expiry date. It should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support .
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion.Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP110 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (substrate buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, polyvalent (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP110) 5 min. 12. Wash with distilled H2O 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, rabbit, rat or guinea pig. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolized by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see also Limitations of the procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3), used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus HRP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus HRP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRPconjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP132) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP133) forms a dark brown precipitate.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 125 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC Single Solution, AEC substrate kit, DAB Substrate kit, DAB High Contrast kit Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Procedure:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP132 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP133 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly.
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus HRP Kits, Mouse is based on the streptavidin-biotin system. It is designed for qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus HRP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus HRP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and horse radish peroxidase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus HRP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Endogenous peroxidase activity in the tissue may cause non-specific staining. This enzyme activity can be blocked by incubation with 3% H2O2-solution (Peroxide Block). Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidin-horse radish peroxidase-conjugate (Streptavidin-HRP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-HRP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the horse radish peroxidase which leads to colour precipitation where the primary antibody is bound. The colour can be observed via a light microscope. The chromogen used determines the colour. The chromogen AEC (included only in kit MON-APP123) leads to the formation of a red-brown product of reaction at the place of the target antigen. The chromogen DAB (included only in kit MON-APP124) forms a dark brown precipitate.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 125 ml Streptavidin-HRP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AEC kit, AEC single solution, AEC substrate kit, DAB substrate kit, DAB high contrast kit. Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O 3% H2O2 solution Reagents for enzyme digestion or heat pre-treatment Wash buffer Pink PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. The tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate AEC working solution (with MON-APP123 only): Add 2 drops (100 µl) of AEC Concentrate to one bottle of AEC Substrate Buffer and mix thoroughly. Preparation of the chromogenic substrate DAB working solution (with MON-APP124 only): Add 4 drops (200 µl) of DAB Concentrate to one bottle of DAB Substrate Buffer and mix thoroughly
Procedure:
1. Peroxide Block (3% H2O2 solution) 10 min. 2. Washing with wash buffer 1 x 2 min. 3. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 4. Washing with wash buffer 1 x 2 min. 5. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 6. Washing with wash buffer 3 x 2 min. 7. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Streptavidin-HRP-Conjugate (Reagent 3, red) 10-15 min. 10. Washing with wash buffer 3 x 2 min. 11. AEC or DAB (Controlling the colour intensity via light microscope is recommended.) 5-15 min. 12. Stopping the reaction with distilled H2O when the desired colour intensity is attained 13. Counterstaining and blueing 14. Mounting: aqueous with AEC, permanent with DAB or Permanent AEC
Expected results:
During the reaction of the substrate with horse radish peroxidase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse. 6. The antigen was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pretreatment or enzyme digestion. If you used a pre-treatment it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. Incubation times were too short or primary antibody concentration too low. 7. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous horse radish peroxidase. Maybe the hydrogen peroxide solution used for blocking was inactivated. 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous peroxidase or pseudoperoxidase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with 3% H2O2 solution. Tissues containing Hepatitis B Surface Antigen (HBsAg) may give false positive results with HRP (horse radish peroxidase) detection systems (Omata et al, 1980). Background staining due to endogenous biotin can be blocked through an avidinbiotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) are available upon request.
The Plus AP Kit, Rabbit is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with mono- and polyclonal primary antibodies and sera obtained from rabbit. The Plus AP Kit, Rabbit can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of the immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Rabbit is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Rabbit binds to rabbit primary antibodies. Therefore this kit can detect mono- and polyclonal primary antibodies and sera obtained from rabbit.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This antibody functions as a link between primary antibody and streptavidin-alkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP128) leads to the formation of a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, Rabbit Reagent 2 (ready-to-use) 125 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit) Permanent AP Red kit, BCIP/NBT Materials required but not supplied: Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP128 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Rabbit (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with AP008RED-RB) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support. No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from rabbit, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific binding.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear.
The Plus AP Kit, Mouse is based on the streptavidin-biotin system. It is designed for the qualitative detection of antigens in fixed paraffin-embedded tissue sections, in frozen tissue sections, and in cytological samples. The kit is developed for use in combination with monoclonal primary antibodies and sera obtained from mice. The Plus AP Kit, Mouse can be used for examining tissues fixed in different solutions, e.g. formalin (neutrally buffered), B5, Bouin, ethanol, or HOPE.
The purpose of immunohistochemical staining is to make tissue and cell antigens visible. The Plus AP Kit, Mouse is a highly sensitive detection kit intended for use in immunohistochemistry and immunocytochemistry. The method is based on the streptavidin-biotin system which means that a biotinylated secondary antibody binds to several molecules of a conjugate composed of streptavidin and alkaline phosphatase. Visualisation occurs via an enzyme-substrate reaction in the presence of a colourising reagent which permits microscopical analysis. The biotinylated secondary antibody in the Plus AP Kit, Mouse binds to mouse primary antibodies. Therefore this kit can detect monoclonal primary antibodies and sera obtained from mice.
Principle of method:
Paraffin-embedded tissue sections are first deparaffinised and rehydrated. Background staining caused by unspecific binding of the primary or secondary antibody is minimized by incubation with a protein blocking solution (Blocking Solution provided with the kit). This step can be omitted if the primary antibodies are diluted in an appropriate buffer. The next step is incubation with the specific primary antibody. After washing, the biotinylated secondary antibody is applied and incubated. This secondary antibody functions as a link between primary antibody and the streptavidinalkaline phosphatase-conjugate (Streptavidin-AP-Conjugate). A second washing is followed by the application of this conjugate. It binds to the biotin at the secondary antibody. Any excess of unbound streptavidin-AP-conjugate is thoroughly washed away after incubation. The addition of the chromogenic substrate starts the enzymatic reaction of the alkaline phosphatase which leads to colour precipitation where the primary antibody is bound. The colour can be observed with a light microscope. The chromogen used determines the colour. The chromogen Permanent Red (included only in kit MON-APP119) leads to a magenta-red product of reaction at the place of the target antigen. Other suitable chromogens are Permanent AP Red (magenta-red) or NBT (blue-black) with its substrate BCIP.
Reagents provided:
125 ml Blocking Solution Reagent 1 (ready-to-use) 125 ml Biotinylated Secondary Antibody, Mouse Reagent 2 (ready-to-use) 125 ml Streptavidin-AP-Conjugate Reagent 3 (ready-to-use) Substrate systems recommended (if not included in the kit): Permanent AP Red Kit, BCIP/NBT Materials required but not supplied Positive und negative control tissue Xylene or suitable substitutes Ethanol, distilled H2O Reagents for enzyme digestion or heat pre-treatment Wash buffer PBS or TBS PAP Pen Primary antibody (user-defined) Primary antibody diluent Negative control reagent Chromogenic substrate Counter stain solution Mounting medium Cover slips
Storage and handling:
The solutions should be stored at 2-8°C without further dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. They should not be used after the expiry date. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents, please contact our technical support
Reagent preparation:
Reagents should be at room temperature when used. Deparaffinise and rehydrate paraffin-embedded tissue sections. Pre-treatment (optional) with HIER (Heat Induced Epitope Retrieval) or enzymatic digestion. Tissue sections have to be completely covered with the different reagents in order to avoid drying out. Preparation of the chromogenic substrate working solution (with MON-APP119 only): Add 2 drops (60 µl) of Permanent Red Concentrate to one bottle of Permanent Red Buffer (Substrate Buffer) and mix. This solution should be used directly after preparation.
Procedure:
1. Blocking Solution (protein block, Reagent 1) (This step is optional.) 5 min. 2. Washing with wash buffer 1 x 2 min. 3. Primary antibody (optimally diluted) or negative control reagent 30-60 min. 4. Washing with wash buffer 3 x 2 min. 5. Biotinylated Secondary Antibody, Mouse (Reagent 2, yellow) 10-15 min. 6. Washing with wash buffer 3 x 2 min. 7. Streptavidin-AP-Conjugate (Reagent 3, red) 10-15 min. 8. Washing with wash buffer 3 x 2 min. 9. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 10. Wash with distilled H2O 1 min. 11. Permanent Red substrate-chromogen solution (with MON-APP119) 5 min. 12. Wash with destilled water 3 x 1 min. 13. Counterstaining and blueing 14. Mounting: aqueous or permanent after dehydration * The incubation times should be adjusted, when using other substrate-chromogen systems.
Expected results:
During the reaction of the substrate with alkaline phosphatase in the presence of a chromogen, a coloured precipitate is formed at the location of the bound primary antibody. This reaction only takes place if the target antigen is existent in the tissue. The chromogen used determines the colour of the precipitate. The analysis is carried out using a light microscope.
Trouble shooting:
If you observe unusual staining or other deviations from the expected results which could possibly be caused by the reagents, please read these instructions carefully, contact our technical support . No staining on an actually positive control slide: 1. Reagents were not used in the proper order. 2. Chromogenic substrate solution was too old. 3. Bleaching because chromogen and mounting medium are incompatible. 4. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. Try a pre-treatment such as heat pre-treatment or enzyme digestion. If you used a pre-treatment it should be extended. 5. Primary antibody not from mouse, but from a different species. 6. The antigen/epitope was not stable in the fixation and/or pre-treatment procedure used. Try another fixation or pre-treatment. Weak staining: 1. Inadequate fixation or overfixation. 2. Incomplete deparaffinisation. 3. The antigen/epitope in the tissue was insufficiently accessible to the primary antibody. If you used heat pre-treatment or enzyme digestion it should be extended. 4. Excessive incubation with Blocking Solution or insufficient washing after this step. 5. Too much wash buffer remains on the slides after washing, diluting the reagents applied in the next step. 6. If you are using PBS-based wash buffer: the activity of alkaline phosphatase in the reagents is blocked if too much wash buffer remains on the slides. 7. Incubation times were too short or primary antibody concentration too low. 8. Chromogenic substrate solution was too old. Non-specific background staining or overstaining: 1. Incomplete deparaffinisation. 2. Excessive tissue adhesive on slides. 3. Insufficient washing especially after the incubation with the enzyme conjugate or the chromogenic substrate solution. These washings are critical. 4. Tissue was allowed to (partially) dry out with reagents on. 5. Unspecific binding of the primary antibody. Please use the Blocking Solution provided with this kit or dilute the primary antibody in appropriate diluents. 6. Incubation time of the primary antibody was too long or primary antibody concentration too high. 7. Incubation time of the chromogenic substrate solution was too long or reaction temperature too high (e.g. if temperature in the laboratory is high). 8. The substrate is metabolised by endogenous alkaline phosphatase in the tissue. This undesired activity can often be suppressed using levamisole (see section Limitations of the Procedure). 9. Non-specific binding of the secondary antibody to endogenous biotin in the tissue section. Carry out an avidin-biotin block before incubation with the primary antibody.
Quality Control:
We recommend carrying out a positive and a negative control with every staining run. The positive control permits the validation of appropriate processing of the sample. If the negative control has a positive result, this points to unspecific staining.
Performance characteristics:
Studies have been conducted to evaluate the performance of the kit reagents. The product has been found to be suitable for the intended use
Limitations of procedure:
Immunohistochemistry is a complex method in which histological as well as immunological detection methods are combined. Tissue processing and handling prior to immunostaining, for example variations in fixation and embedding or the inherent nature of the tissue can cause inconsistent results (Nadji and Morales, 1983). Endogenous alkaline phosphatase activity or the endogenous biotin content may cause non-specific staining. The enzyme activity can be blocked by incubation with levamisole. However, neither intestinal nor placental alkaline phosphatase can be blocked with levamisole. Background staining due to endogenous biotin can be blocked through an avidin-biotin blocking step prior to the primary antibody incubation step. Inadequate counterstaining and mounting can influence the interpretation of the results. The colour intensity of the reaction product can decrease with time, especially when exposed to light. Overexposure with the protein blocking solution (Blocking Solution) can result in decreasing signal intensity. Therefore, we recommend washing away the Blocking Solution instead of just draining it away as in other procedures. Sanbio guarantees that the product will meet all requirements described from its shipping date until its expiry date, as long as the product is correctly stored and utilized. No additional guarantees can be given. Under no circumstances shall Sanbio be liable for any damages arising out of the use of the reagent provided.
Precautions:
Use by qualified personnel only. Wear protective clothing to avoid eye, skin or mucous membrane contact with the reagents. In case of a reagent coming into contact with a sensitive area, wash the area with large amounts of water. ProClin 300 and sodium azide (NaN3) are used for stabilisation. Sodium azide deposits in drainage pipes made of lead or copper can result in the formation of highly explosive metallic azides. To avoid such deposits in drainage pipes, sodium azide should be discarded in a large volume of running water. Material safety data sheets (MSDS) for the pure substances are available upon request. Microbial contamination of the reagents must be avoided, since otherwise non-specific staining might appear
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