It is a research based on the principle of antigen and antibody specific binding，to make the chromogenic reagent (enzyme) color of the labeled antibody by chemical reaction，to determine the intracellular antigen (polypeptide and protein)，and make the position, qualitative and quantitative analysis.
1.1 Deparaffinize and rehydrate: incubate sections in 3 changes of xylene, 15 min each. Dehydrate in 2 changes of pure ethanol for 5 min, followed by dehydrate in gradient ethanol of 85% and 75% ethanol, respectively, 5 min each. Wash in distilled water.
1.2 Antigen retrieval: immerse the slides in Sodium citrate antigen retrieval solution (pH 6.0) and maintain at a sub-boiling temperature for 8 min, standing for 8 min and then followed by another sub-boiling temperature for 7 min. Be sure to prevent buffer solution evaporation. Let air cooling. Wash three times with PBS (pH 7.4) in a shaker device, 5 min each.
1.3 Block endogenous peroxidase: wash three times with PBS (pH 7.4) in a shaker device, 5 min each. Immerse in 3% H2O2 and incubate at room temperature for 15 min, kept in dark place. Then wash again three times with PBS (pH 7.4) in a Rocker device, 5 min each.
1.4 Block with serum: eliminate obvious liquid, mark the objective tissue with liquid blocker pen. Cover objective tissues with 10% normal rabbit serum (for the case of primary antibody originated from goat) or 3% BSA (for the case of primary antibody originated from others) at room temperature for 30 min.
1.5 Primary antibody: throw away the blocking solution slightly. Incubate slides with primary antibody (diluted with PBS appropriately) overnight at 4 ℃, placed in a wet box containing a little water.
1.6 Secondary antibody: wash slides three times with PBS (pH 7.4) in a shaker device, 5 min each. Then throw away liquid slightly. Cover objective tissue with secondary antibody (appropriately respond to primary antibody in species) labelled with HRP, incubate at room temperature for 50 min.
1.7 DAB developing: dry sections slightly, add fresh prepared DAB chromogenic reagent to marked tissue. Manage reaction time by observing in microscopy until nucleus shows brown-yellow. Then stop developing reaction by wash in running tap water.
1.8 Counterstain in nucleus with Hematoxylin staining solution for 3 min and wash in tap water. Treat with the differentiate solution for a few seconds, wash in running tap water. Back to blue by bluing solution, wash in running tap water.
1.9 Dehydrate successively in gradient ethanol of 75%, 85%, and 2 changes of pure ethanol, respectively, 6 min each. Clear in xylene for 5 min and mount with resin mounting medium.
Nucleus stained with hematoxylin are blue. The positive cells developed by DAB reagent have brown-yellow nucleus.