Immunofluorescence Staining for Oxidative Stress and Apoptosis

Immunofluorescence staining was performed according to standard protocols (Sun et al., 2020 (link); Li et al., 2021 (link)). The paraffin embedded heart sections and cell slides were incubated with the following primary antibodies at 4C overnight: goat anti-8-hydroxy-2′-deoxyguanosine (8-OHdG) (Sigma, Cat. # AB5830, 1:500 dilution) and rabbit anti-caspase-3 antibody (Abcam, Cat. # ab32351, 1:500 dilution). After washing, sections were incubated with donkey anti-goat IgG 647 (Abcam, Cat. # ab150135, 1:1,000 dilution) or goat anti-rabbit IgG 555 (Abcam, Cat. # ab150078, 1:1,000 dilution) at room temperature for 2 h, and counterstained with 1 μg/ml DAPI. All fluorescence images were obtained using the Nikon Eclipse Ti-U fluorescence microscope and analyzed from five randomly selected fields by ImageJ.