TUNEL assay was performed according to the standard protocol (R&D system). Briefly, slides were baked for 60 min at 60°C, deparaffinized, and rehydrated through xylene and graded alcohol incubation. Tissues were pretreated with protein digestion enzyme or proteinase K (20 μg/ml) for 15 min, washed, and quenched in 3% H2O2 in PBS. Equilibration buffer was applied for 10 s, followed by incubation with 55 μl of TdT enzyme in a humidified chamber at 37°C for 1 hour. Stop/wash buffer and anti-digoxigen conjugate were subsequently applied, and color was developed in peroxidase substrate before washing in a coplin jar. Counterstaining was performed with hematoxylin and 0.25% HCl-alcohol before dehydration and mounting for visualization. TUNEL slides were scanned by Aperio ScanScope and divided into four quadrants in the software; two of eight random rectangular regions of size 500 μm × 500 μm (0.25 mm2) were selected and counted for positively stained cells. The investigator was blinded to the group and labeling allocation during the experiment.

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