2.4. TUNEL Assay in Heart Tissue

Cardiac apoptotic nuclei were performed with a Situ Cell Death Detection Kit (Roche, Indianapolis, IN, USA) assay according to the manufacturer’s instructions. In brief, the sections were treated with proteinase K, followed by permeabilization solution, a blocking buffer. A TUNEL and fluorescein isothiocyanate-dUTP assay proceeded for 60 min at 37 °C. Finally, all of the slides were mounted using a vectashield with 4,6-diamidino-2-phenylindole (DAPI). The number of TUNEL-positive cardiomyocytes was examined using a fluorescent microscope (DP 74, Olympus, Tokyo, Japan).