TUNEL Assay for Apoptosis Detection

Tunnel assay was performed according to DeadEnd™ Fluorometric TUNEL System (Promaga, Tokyo, Japan) manufacturer’s instructions. Briefly, tumor sections were treated with fresh xylene in a Coplin jar for 5 min twice at room temperature. Rehydrate the samples by sequentially immersing the slides through graded washes (100%, 95%, 85%, 70%, 50%, 0.85% NaCl, PBS) for 5 min. The tissue sections were fixed by immersing the slides in 4% methanol-free formaldehyde solution in PBS for 15 min. After washed in PBS for 5 min three times, the samples were incubated with 100 µl proteinase K (20 µg/ml) for 8–10 min. Then were washed and incubated with 100 µl Equilibration Buffer for 5-10 min. The tissue sections were fixed with 4% methanol-free formaldehyde solution in PBS for 5 min. Then washed and incubated with 50 µl of rTdT incubation buffer at 37 °C for 60 min in the dark. The reactions were terminated by incubated with 2X SSC for 15 min at room temperature. The samples were washed three times and were stained by propidium (1 µg/ml) in PBS for 15 min at room temperature in the dark. Then the samples were washed three times and analyzed by the Olympus FV1000 IX81-SIM Confocal Microscope (Olympus, Tokyo, Japan).