IHC Protocol of SABC and SV Methods

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.

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.

• 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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