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 3 times of xylene, 15 min each time. Dehydrate in 2 times of pure ethanol for 5 min, followed by dehydrating in gradient ethanol of 85% and 75% ethanol respectively, 5 min each time. 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 evaporate or water evaporation. Wash three times with PBS (pH 7.4) in shaker, 5 min each time.
1.3 Block endogenous peroxidase: wash three times with PBS (pH 7.4) in shaker, 5 min each time. Immerse in 3% H2O2 and incubate at room temperature for 25 min, keeping in dark place. Then wash again three times with PBS (pH 7.4) in shaker, 5 min each time.
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 °C , placed in a wet box containing a little water.
1.6 Secondary antibody: wash slides three times with PBS (pH 7.4) in shaker ,5 min each time. 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: wash slides three times with PBS (pH 7.4) in shaker, 5 min each time. 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 times of pure ethanol, respectively, 6 min each time. Clear in xylene for 5 min and mount with neutral gum.
Nucleus stained with hematoxylin are blue. The positive cells developed by DAB reagent have brown-yellow nucleus.