2.6.2 Immunohistochemical Staining SABC Method

2.6.2 Immunohistochemical Staining (SABC Method)

Wash the sections with PBS for 5 minutes each time, three times. Add 3% H₂O₂ and let it sit at room temperature for 10 minutes to eliminate endogenous peroxidase activity.

Wash the sections with PBS for 5 minutes each time, three times. Antigen retrieval: Place the sections in a 0.01 M sodium citrate buffer solution (pH 6.0), heat in a water bath to about 92–95°C, boil for 15 minutes, then let cool naturally for 20–30 minutes before taking out the sections.

Wash the sections with PBS (pH 7.2–7.6) for 3 minutes each time, three times.

Add normal goat serum blocking solution and block at room temperature for 15 minutes, then shake off excess liquid (do not wash).

Add primary antibodies (rabbit anti-mouse P27 monoclonal antibody, rabbit anti-mouse Bcl-2 monoclonal antibody) and let stand at 37°C for about 1 hour. Wash the sections with PBS (pH 7.2–7.6) for 3 minutes each time, three times.

Add biotinylated secondary antibody working solution (biotinylated goat anti-rabbit IgG) and let stand at 37°C for 15 minutes. Wash the sections with PBS for 3 minutes each time, three times.

Add horseradish peroxidase-labeled streptavidin working solution and incubate at 37°C for 15 minutes. Wash the sections with PBS for 3 minutes each time, three times.

DAB staining: Use a DAB staining kit. Take 1 mL of distilled water, add one drop each of reagents A, B, and C from the kit, mix well, and apply to the sections. Let it stain at room temperature, control the reaction time under a microscope (sections with the same indicators should have the same staining reaction time to avoid staining errors), generally between 5 and 30 minutes. Rinse with distilled water.

Lightly counterstain with hematoxylin.

Stop the reaction by rinsing with tap water.

Dehydrate step by step, make transparent with xylene, seal with neutral gum, and observe the expression of tissue indicators under a microscope.

---

Research on Pei Zhengxue’s series of prescriptions