TUNEL Detection of Apoptosis

Apoptosis of the tumor cells following treatments in vivo was determined by TUNEL staining using a commercially available kit (In Situ Cell Death Detection Kit, POD; Roche, Germany). Tumor sections were deparaffinized and dehydrated according to standard protocols. Tissue sections were incubated with Proteinase K working solution for 30 minutes at 21°C to 37°C. The slides were then washed twice with PBS (pH 7.2-7.6). The positive control was incubated with DNase I for 10 minutes at 15°C to 25°C, and the negative control was incubated with label solution (without terminal transferase) instead of the TUNEL reaction mixture. The slides were then washed three times with PBS (pH 7.2-7.6). Converter-POD was added to the slides which were then incubated in a humidified chamber for 30 minutes at 37°C. Following this, the slides were washed three times with PBS (pH 7.2-7.6). The 3,3-diaminobenzidine substrate was added onto the slides and incubated for 10 minutes at 15°C to 25°C, after which they were then washed three times with PBS (pH 7.2-7.6). The slides were then mounted and analyzed through the microscope (Olympus, Melville, NY, USA). The slides were viewed at 400× magnification, and apoptotic cells were identified by the brown-stained appearance of the cells. Expression levels were quantified based on the average optical density (AOD) of the positive cells in five fields/sample using the Image-Pro Plus 6.0 software.