4.7. In Situ Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay and Evaluation of Ki-67 Proliferation

Nuclear DNA fragmentation in tumor tissues was detected using a DeadEnd™ Fluorometric TUNEL System according to the manufacturer’s instructions. Briefly, paraffin-embedded sections were deparaffinized and hydrated in a series of graded ethanols and then digested with trypsin for 40 min at room temperature. The tissue sections were then incubated with TUNEL reaction buffer in a 37 °C humidified atmosphere for 60 min, washed with PBS, and then incubated with DAPI for 1 min at room temperature. Tumor proliferation was identified by anti-Ki-67 antibody staining according to the manufacturer’s protocol. Slides were visualized under a fluorescence microscope (OLYMPUS CX41, Tokyo, Japan). TUNEL-positive or Ki-67-positive cells were counted at ×400 magnification. The apoptotic index or proliferation index was then defined as a ratio of (the number of apoptotic TUNEL-positive or Ki-67-positive cells)/(the total number of cells) in each field.