Quickblock immunostaining blocking solution

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.

Mice were anesthetized 48 h after reperfusion, transcardially perfused with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde. Their brains were rapidly removed and postfixed with 4% paraformaldehyde. Brains were then immersed in 20% and 30% sucrose overnight at 4°C, embedded in Tissue-Tek OCT compound (Sakura, Japan), and subjected to section on a freezing microtome. The sections (10 μm) were washed with PBS and blocked in QuickBlock™ immunostaining blocking solution (Beyotime, China) for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: rabbit anti-Sertad1 (1:200, ARP34309_T100; AVIVA, USA), mouse anti-NeuN (1:100, 66836-1-Ig; Proteintech, China), mouse anti-GFAP (1:200, ab279290; Abcam, USA), mouse anti-Iba1 (1:500, GT10312; GeneTex, USA). After washing, the sections were incubated with Alexa Fluor Plus 488 goat anti-rabbit secondary antibody (1:200, A32731; Thermo Fisher Scientific, USA) or Alexa Fluor Plus 555 goat anti-mouse secondary antibody (1:200, A32727; Thermo Fisher Scientific) at 37°C for 1 h. DAPI (Beyotime) was used to stain cellular nuclei at 37°C for 10 min. Finally, the sections were photographed using a microscope with a digital camera (Olympus, Japan).