*2.7. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL Assay)*

To identify DNA fragmentation, we performed TUNEL staining using an ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Intergen Company, Purchase, NY, USA), according to the manufacturer's instructions. The slides were soaked in an ethanol-acetic acid solution (2:1 v/v dilution), placed in a freezer for 5 min, and washed thrice with PBS. Sections were sequentially incubated with 0.5% Triton X-100, Protease K (100 mg/mL), 3% hydrogen peroxide, and TUNEL reaction mixture, which was used to rinse the samples after each step. The staining was performed using horseradish peroxidase-tagged antibodies and 0.03% diaminobenzidine, with counterstaining using the Nissl dye. TUNEL-positive myonuclei were counted in two sections of LV in 4 rats per group, and the averages were calculated as percentages of the total number of labeled myonuclei.
