Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) staining

TUNEL staining was performed using a commercial cell death detection kit (Roche Diagnostics) according to the manufacturer's protocols. Briefly, after deparaffinization and rehydration, the slices were pretreated by proteinase K (PCR grade; Roche Diagnostics) for 15 min at room temperature. Then, the permeabilization was conducted with fresh prepared 0.1% Triton-X-100 and 0.1% sodium citrate for 8 min at room temperature. Then, incubation with trypsin (0.01 N HCL) was applied for 30 min at 37°C. Subsequently, the slices were transferred into a container with 0.1 M citrate buffer (pH, 6) and received microwave irradiation (350 W) for 5 min. Rinsed slices were treated with TUNEL mixture for 60 min at 37°C in a humidified atmosphere in the dark. Finally, the signal conversion was handled with the help of converter-POD and DAB substrate. The stained slices were observed by a light microscope (magnification, ×400; DM2500; Leica Microsystems GmbH) and images were captured. The index of apoptosis was calculated as the percentage of apoptotic cells among all cells counted. Cells with dark brown-stained nuclei were counted by two independent investigators who were blinded to the group assignments. At least three visual fields per slide and five slides per group were evaluated by the investigators.