Myocardial apoptosis was performed as previously described (34) with minor modifications. Sham- or IR-operated mice were euthanized 4 hours after reperfusion, and hearts were quickly removed and fixed in 4% paraformaldehyde. The hearts were then exposed to sucrose and cryofixed. Hearts were then sectioned on a cryostat with sections measuring 5 μm in thickness. TUNEL staining was carried out with the In Situ Cell Death Detection Kit as per the manufacturer’s protocol (Roche). Slides were counterstained with α-actinin (Sigma A7811) and DAPI to label cardiomyocytes and nuclei, respectively. Slides were mounted with Fluoromount-G (Southern Biotech), and images were acquired with a 20× objective on a Nikon TiE fluorescence microscope. The apoptotic index was determined by using NIS-Elements Software (Nikon, Japan). On average, about 7000 cells were counted per heart.

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