1.1 Deparaffinize and rehydrate: incubate sections in 2 cylinders of xylene for 20 min each. Dehydrate in 2 cylinders of pure ethanol for 10 min each, followed by dehydrating in gradient ethanol of 95%, 90%, 80%, and 70% ethanol, respectively 5 min each time (extend deparaffinization time slightly in winter). Wash in distilled water.
1.2 Antigen retrieval: eliminate obvious liquid, mark the objective tissue with marker pen. Add proteinase K working solution to cover objectives and incubate at 37°C for 25 minth, then wash three times with PBS (pH 7.4) in shaker, 5 min each time.
1.3 Permeabilization: eliminate excess liquid, add permeabilize working solution to cover objective tissue, then incubate at room temperature for 20 min. Wash three times with PBS (pH 7.4) in shaker, 5 min each time.
1.4 Tunel reaction: mix reagent 1 (TdT) and reagent 2 (dUTP) (both from the Tunel assay kit) at ration of 1:9. Prepare this reaction solution according to demand before use. Add this mixture to objective tissue placed in a flat wet box, incubate at 37°C for 2 hour. Make sure to keep the wet box moist by adding water.
1.5 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 15 min, keeping in dark place. Then wash again for three times, 5 min each time.
1.6 Detection reaction: dry sections slightly. Cover tissues with reagent 3 (converter-POD) and incubate at 37°C for 30 min, keeping in a wet box in dark place. Then wash three times with PBS (pH 7.4) in shaker, 5 min each time.
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 washing 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 70%, 80%, 90% and 95%, 5 min each time. Then dehydrate in 2 cylinders of pure ethanol. Clear in 2 cylinders of xylene. Dry briefly and mount with resin mounting medium.
Nucleus stained with hematoxylin are blue. The positive apoptosis cells developed by DAB reagent have brown-yellow nucleus.