First Edition (Revised on April, 2016)
SABC (Strept Avidin Biotin-peroxidase Complex Method), is a immunochemistry staining method for displaying the distribution of antigens in tissues and cells. Streptavidin is a tetrameric protein purified from the bacterium Streptomyces avidinii. Streptavidin is a heterocycle monocarboxylic acid contains a S element. It labels antibody by the combination of its carboxyl and the amidogen of the protein. A Streptavidin has extraordinarily strong affinity to biotin molecules, and One tetrameric molecule of streptavidin can combine with four molecules of biotin. Streptavidin has very low non-specific binding to tissues and cells, therefore, immunohistochemical analyses based on streptavidin-biotin complex has extremely low background. Furthermore, this kit has high sensitivity because each complex it generates has a large number of peroxidase and streptavidin molecules. In brief, SABC offers high specificity, low background and ease-of-use.The kit is designed for displaying the distribution of antigens in immunochemistry, and it’s suitable for paraffin sections, frozen sections, cultured cells and fresh prepared blood smears.
|Reagents||Amounts||Ingredients and usage|
|Normal cavia serum blocking reagent||10mL||Block of tissue sections|
|Biotinylated Secondary Antibody||0.2mL(50×)||Cavia Anti-rabbit IgG|
|SABC||0.2mL(50×)||Peroxidase conjugated streptavidin|
STORAGE AND PERIOD OF VALIDITY
4℃ for one year. Avoid freezing.
MATERIAL REQUIRED BUT NOT PROVIDED
1. APES or POLY-L-LYSINE.
2. 0.02M PBS(pH7.2-pH7.6): Na2HPO4·12H2O 7g, NaH2PO4·2H2O 0.5g, NaCl 9g in 1L of distilled water.
3. 0.01M Citrate Buffer: 3g sodium citrate dihydrate (C6H5Na3O7·2H2O) and 0.4g citric acid monohydrate (C6H8O7·H2O) in 1L of distilled water.
4. DAB Chromogen Kit.
IMMUNOHISTOCHEMISTRY IN PARAFFIN SECTION
1. Cover the entire surface of a clean microslide with APES or POLY-LLYSINE, dry at room temperature or bake in an oven at 60℃for one hour.
2. Dewax the tissue section, and rinse with water.
3. Incubate the tissue section for 5~10 minutes in the 3% H2O2 solution to quench the endogenous peroxidase activity.
4. Wash the tissue section with distilled water 3 times.
5. Select a proper method to repair antigen. The characteristics of the antigen used may be used as a guideline.
Microwave oven heat repair antigen process in paraffin sections: soak the tissue section in 0.01M citrate buffer (pH6.0), and heat to the boiling point with an electric heater or a microwave oven, then stop heating. Repeat this heating process 1~2 times with a 5~10-minute interval. Wash the tissue section with 0.02M PBS (pH7.2~7.6) once or twice when it cools at room temperature.
High pressure repair antigen process in paraffin sections: soak the tissue section in 0.01M citrate buffer(pH6.0), lid the pot cover, when the pressure cooker begins to deflate(about 5~6 minutes), remove the heat source after 1~2 minutes. Take down the air valve, open the pot cover, and take out the section after it cools at room temperature. After wash it with distilled water, then wash with PBS 3 times.
Enzyme digestion process: Incubate the tissue section in 0.1% trypsinase at 37℃ for 10~40 minutes to expose intracellular antigens. or use 0.4% trypsinase at 37℃ for 30~180 minutes to expose intercellular antigens. 6. Add normal cavia serum blocking reagent to the tissue section and incubate at room temperature for 20 minutes. Discard the blocking reagent solution, but do not wash the tissue section.
7. Add properly diluted primary antibody (rabbit IgG) to the tissue section and incubate at 37°C for about 1 hour or 20°C for about 2 hours or at 4°C overnight.
8. Wash the tissue section with PBS (pH 7.2~7.6) 3 times for 2 minutes each.
9. Add biotinylated cavia anti-rabbit IgG to the tissue section and incubate at 20~37°C for 20 minutes. Wash the tissue section with PBS (pH 7.2~7.6) 3 times for 2 minutes each.
10. Add SABC-Peroxidase (Streptavidin-Peroxidase) to the tissue section and incubate at 20~37°C for 20 minutes. Wash the tissue section 4 times with 0.02M PBS (pH 7.2~7.6) for 5 minutes each.
11.Use a DAB chromogenic kit (Catalog number: USCNdab01) to stain the tissue section. Mix Reagent A, B thoroughly as the manual, then add appropriate volume to the tissue section and incubate at room temperature. Observe under the microscope about 5~10 minutes. When reach a proper color intensity(high positive staining, weak background), Wash the tissue section with distilled water to end the reaction.
12. Slightly counterstain the tissue section with haematoxylin .Then dry the tissue section, and put on a drop of resin seal the tissue section with a cover slide. The tissue section is ready for observation under a microscope.
1. The primary antibody concentration, incubation time and temperature directly affect the staining efficiency and background intensity. If the positive staining is too weak, the concentration of the primary antibody and the incubation time can be increased; if the background is too high, the primary antibody concentration and the incubation time can be decreased. Such as wash the section with 0.01-0.02% TWEEN 20 PBS 4 times and with pure PBS twice after SABC reaction and before DAB staining, then use DAB chromogenic kit to stain the section.
2. PBS, TBS and other buffers can be used instead of the 0.01 M Citrate Buffer (pH6.0)in heat repair antigen process.
3. Dewax the tissue section thoroughly to avoid non-specificity background staining occurred. It’s recommended to separate the dewaxing in immunochemistry and in the H.E.
4. Take special measures of the tissue section in blocking when the kit is used for liver or kidney, which are rich in endogenous avidin-bindingacticity.
5. All reagents should be used within the expiration date of the kit, and kits from different batches not be used cross.
Warning: As DAB is possibly carcinogenic, so take necessary measures when use it.