IHC Protocol for Mouse Tissue Sections

Immunohistochemistry is the study of localization, characterization and quantification of antigens (peptides and proteins) in tissue cells by applying the basic principle of immunology antigen-antibody reaction, i.e., the principle of specific binding of antigens and antibodies, through chemical reactions to develop color of chromogenic agents (fluorescein, enzymes, metal ions, isotopes) that label antibodies, called immunohistochemistry techniques.

• Paraffin slices were baked overnight in a 60°C oven.
• Dewax in xylene, gradient alcohol into water (anhydrous ethanol, 95% alcohol) and soak in distilled water to be used.
• Antigen repair. Take 500 ml of EDTA antigen repair working solution in a 1000 ml beaker and heat it on a low power electric stove until it seems to boil slightly (to prevent desquamation). Place the tissue sections slowly into the beaker. Continue heating, keeping the liquid at a slight boil for 20 minutes. Remove the beaker from the heat and remove the slices after natural cooling at room temperature, wash once with distilled water for 3 minutes and twice with TBS for 3 minutes each.
• Add one drop or 50 μL of 3% hydrogen peroxide solution to each section and incubate for 10 min at room temperature to block the endogenous peroxidase activity. Rinse 3 times with TBS for 3 min each time.

• Remove the TBS solution, add one drop or 50ul of primary antibody (multiple antibodies obtained from inactivated PLB immunized mice, diluted 1:3000), and incubate the negative control with normal serum for 2 hours at room temperature, or overnight at 4°C.
• Wash 3 times with TBS for 5 min each time, remove the TBS solution, add one drop or 50 μL of polymer enhancer (Reagent A) to each section, incubate for 20 min at room temperature, rinse 3 times with TBS for 3 min each time.
• Remove the TBS solution, add one drop or 50 μL of peroxidase-labeled anti-mouse/rabbit polymer (Reagent B) to each section, incubate for 30 min at room temperature, and rinse three times with TBS for 3 min each time.
• Remove TBS solution, add one drop or 50ul of freshly prepared DAB to each section and develop color for 3-10 min.
• Rinse with tap water, hematoxylin re-staining for 10min, rinse with water. 0.1% hydrochloric acid fractionation, rinse with tap water, return to blue with TBS.
• Without dehydration, directly seal the film with neutral resin.

Antigen repair must be performed in such a way that the section can always be immersed in the liquid and must wait for the working solution to cool before removing the section.

Immunohistochemistry (IHC), Immunofluorescence (IF) Service

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