TUNEL staining for apoptosis

Tumor cell apoptosis was detected using the In-Situ Cell Death Detection Kit, TMR red (Roche, Basel, Switzerland) with TUNEL technology, in accordance with the manufacturer’s instructions. The dewaxed sections were permeabilized with Proteinase K working solution (20μg/ml, provided in the kit) at 37˚C for 30 min. After being washed 3 times in PBS for 5 min each time, the sections were incubated with 50 μl of TUNEL reaction mixture (provided in the kit) in a dark and humid atmosphere for 60 min at 37˚C and then washed 3 times in PBS for 5 min each time. Subsequently, the sections were incubated in the dark for 5 min with DAPI (Beyotime, Shanghai, China) to stain the nucleus and then washed 4 times in PBST for 5 min each time. Finally, the sections were sealed with anti-fluorescence quenching agent (Southern Biotech, Alabama, USA) and then analyzed under a BX53 fluorescence microscope (Olympus Co., Tokyo, Japan). For quantification of TUNEL-positive cells, the number of TUNEL-positive and total tumor cells was counted in 3 random fields (×400 magnification). The apoptosis index (AI) was calculated as the ratio of TUNEL-positive to total tumor cells. The ratio of AI (R_AI) was calculated on basis of R_AI=left AI/right AI.