Apoptosis of heart sections and cardiomyocytes was detected using an In Situ Cell Death Detection Kit (TUNEL fluorescence FITC kit; Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. The heart sections were stained with a commercially available TUNEL kit. Nuclei were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA). To identify cardiomyocytes, counterstaining by primary antibody a-actinin (Sigma-Aldrich) was performed after TUNEL staining. Fluorescence staining was viewed by microscopy (Nikon). Six to eight visual fields were randomly selected from each section. The percentage of TUNEL-positive nuclei was calculated as the sum of all double-positive nuclei divided by the sum of all nuclei.

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