Fluoview fv300 version 3c acquisition software

Heart sections were deparaffinized and rehydrated using a gradient elution method with xylene and ethanol. Antigen retrieval was performed by incubation in citrate antigen retrieval solution (P0081, Beyotime) at 95 °C for 20 min. The cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The prepared heart sections and cells were blocked by incubation in QuickBlock™ Immunostaining Blocking Solution (P0260, Beyotime) at 37 °C for 30 min, followed by overnight incubation at 4 °C with primary antibodies against γ-H2AX (1:200, ab81299, Abcam, Cambridge, MA), cardiac troponin T (1:200, ab209813, Abcam), and BNIP3 (1:200, ab109362, Abcam). The slides were then incubated with Alexa Fluor 488™ secondary antibody (1:200, A-11008, Thermo Fisher Scientific) or AlexaFluor555™ secondary antibody (1:200, A-11035, Thermo Fisher Scientific) in the dark at 37 °C for 1 h. The nucleus was counterstained with DAPI (C1006, Beyotime, China). Each step was followed by washing with PBS three times for 5 min. The images were captured using laser confocal microscopy and analyzed with the Olympus Fluoview FV300 version 3C Acquisition Software. Quantitative analysis of the fluorescence intensity was conducted in a blinded manner using Image J software.