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IHC Protocol of SABC and SV Methods
GUIDELINE
The Strept Avidin-Biotin Complex (SABC) method is a simple and sensitive immunohistochemical method for displaying antigen distribution in tissues and cells. The SV (Super Vision) two-step method uses a polymerization labeling method to link peroxidase with secondary antibodies to form a super-sized antibody-enzyme polymer, replacing secondary and tertiary antibodies in the traditional method. This polymer possesses a powerful signal amplification ability and also has a strong tissue cell penetration. It has been tested to be more sensitive than similar products, especially against nuclear antigens, and its permeability is particularly strong for immunohistochemical applications.
METHODS
SABC Method
- Dewaxing of slices to water.
- Fresh configuration of 3% H2O2 at room temperature for 10 minutes to inactivate endogenous enzymes, followed by rinsing with distilled water for 2 minutes three times.
- Microwave repair. Place the slices in a cup containing PBS buffer (PH 7.2-7.6), heat and boil in a microwave oven, let stand in the microwave for 5 minutes, take out and cool at room temperature for 6 minutes, then microwave renewed heat to boiling and cool completely at room temperature.
- Add 5% BSA closure solution dropwise and react at 37°C for 30 minutes. Shake off excess liquid without washing.
- Add primary antibody dropwise and react overnight at 4°C. The concentration of the primary antibody prepared is 1 µg/ml.
- Wash in PBS buffer (pH 7.2-7.6) for 10 min x 3 times, add biotinylated secondary antibody IgG (anti-rabbit or anti-mouse) dropwise and react at 37°C for 30 min.
- Wash in PBS buffer (pH 7.2-7.6) for 10 min x 3 times, dropwise addition of reagent SABC, 30 min reaction at 37°C, 30 min PBS wash x 3 times.
- Color development of DAB at room temperature, control reaction time under the microscope, then wash with distilled water
- Add hematoxylin dropwise for 60 seconds for light re-staining, wash with distilled water and then immerse in saturated Na2HPO4 solution for 2 minutes, remove and wash, dry overnight at 37°C thermostat and seal the film with neutral gum.
SV Method
- The first 5 steps are the same as the SABC method.
- Wash with PBS buffer (pH 7.2-7.6) for 10 min x 3 times, add HRP-labeled secondary antibody IgG (anti-rabbit or anti-mouse) dropwise for 30 min at 37°C and wash with PBS buffer (pH 7.2-7.6) for 30 min x 3 times.
- The last 2 steps are also the same as the SABC method.
NOTES
- The process of dewaxing should be done in a fume hood at room temperature in summer. When the temperature is lower than 18°C, it is recommended to dewax at 50°C.
- If the staining background is too high, wash the section 4X with 0.01-0.02% TWEEN 20 PBS and 2X with pure PBS after the SABC reaction and before DAB staining. Then use DAB to stain the samples.
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