Immunohistochemistry Analysis

Heart tissue sections were deparaffinized with dimethylbenzene, dehydrated using an alcohol gradient, and incubated with 3% H₂O₂ to block endogenous peroxidase and nonspecific binding sites. Afterward, sections were incubated with corresponding primary antibodies overnight at 4°C (Nrf2, 1:50; BAX, 1:50; Bcl-2, 1:50; caspase3, 1:50; p-ROS, 1:50). Heart tissues were observed under a microscope (400 × magnification, Eclipse E100 + Eclipse FN1, Nikon, Tokyo, Japan) using six randomly selected fields, followed by analysis using Image-Pro Plus image analysis software (Media Cybernetics, Inc. Rockville, MD, United States).