Immunohistochemistry and TUNEL staining

The rat heart tissues were fixed with 4% paraformaldehyde. After 24 hours, the tissues were embedded in paraffin and cut into sections. Xylene was used to separate the paraffinized sections before they were rehydrated using gradient ethanol. Then, antigen extraction was carried out with 10 mM citric acid buffer, and the tissue sections were incubated in 3% H₂O₂ for 10 minutes and sealed at room temperature for 1 hour. Subsequently, the sections were subjected to overnight incubation with rabbit anti-caspase-3 antibody (1:1,000, #9662, Cell Signaling). The corresponding second antibody was incubated at room temperature for 1 hour. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was carried out to measure cell apoptosis in situ in accordance with the instructions of the manufacturer (TUNEL Apoptosis detection kit: UPSTATE, Lake Placid, NY, USA). Apoptotic cells were defined as those with nuclei stained yellowish-brown. Images were captured with a special OLYMPUS DX51 fluorescence microscope (Tokyo, Japan). The data were analyzed by image 6.0.