TUNEL Apoptosis Assay

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) detection kit (Promega, USA) was employed for the detection of cell apoptosis. Briefly, paraffin-embedded sections were deparaffinized and dehydrated. Sections were treated with 20 μg/ml Proteinase K for 20 min, rinsed with 0.3% Triton X-100 for 10 min, incubated with TUNEL reaction mixture at 37 ^oC for 1 h, treated with HRP conjugated streptavidin (1:200; Beijing Zhongshan Biotech, China) at 37 ^oC for 30 min, treated with 0.04% DAB and 0.03% H₂O₂ at room temperature for visualization for 8-12 min, counter stained with hematoxylin. In the interval of experiment steps washing in PBS three times (3 min for each). In the negative control, TUNEL reaction mixture was replaced with PBS. The positive control sections were pre-treated with DNase I for 10 min followed by TUNEL staining. Cells with blue granules in the nucleus were regarded as positive for TUNEL.