Detection of apoptosis using a TUNEL assay

Cell apoptosis was detected using a TUNEL apoptosis detection kit (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's protocol. In brief, the paraffin-embedded sections of gastric cancer tissues were dewaxed at 60°C, washed twice with xylene for 5 min each time, rehydrated in a descending alcohol series (100, 95, 90, 80 and 70% for 5 min of each alcohol concentration) and processed with proteinase K at 37°C for 20 min, followed by processing with 0.1% Triton X-100 for 8 min and rinsing with phosphate-buffered saline (PBS) twice, for 5 min each time. After incubation with TUNEL reaction mixture at 37°C in the dark for 60 min, the sections were washed with PBS 3 times (5 min/wash). The sections were further counterstained with 5 µg/ml propidium iodide (PI) dye at room temperature for 5 min and mounted with gum. The number of apoptotic cells per 100 cells in each field of view of five random fields was counted under an inverted fluorescence microscope (magnification, ×400; Nikon Corporation, Japan). The apoptosis index (AI) was calculated as follows: AI=(the number of apoptotic cells/total cell count) ×100%.