4 6 diamidino 2 phenylindole dapi

Frozen aortic roots sections or macrophages were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.2% Triton X‐100 for 15 min. After blocking with 10% goat serum, the tissue sections or macrophages were indicated with primary antibodies at 4°C overnight. After washing with PBS for 3 times, tissue sections or macrophages were incubated with fluorescein‐conjugated secondary antibodies (Santa Cruz Biotechnology) for 2 h at room temperature. The cell nuclei were stained by incubation with 4, 6‐diamidino‐2‐phenylindole (DAPI, Bioss, Woburn, MA, USA). The fluorescence was detected and photographed by confocal microscopy (Olympus, Tokyo, Japan). The fluorescence intensity was quantified with ImageJ software, and relative fluorescence intensity was calculated as the targeted fluorescence intensity relative to the DAPI fluorescence intensity. Besides, the co‐localization analysis was performed using co‐localization plug‐in for ImageJ after converted the individual fluorescent channel images to 8‐bit grayscale.

The A549 cells were grown on Nunc™ Petri dish (Thermo Fisher, Waltham, MA, USA) and then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) buffer for 30 min. After washing with PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS buffer for 15 min and then blocked with 1% bovine serum albumin (BSA) (Thermo Fisher, Waltham, MA, USA) for 30 min. After blocking, the cells were incubated (4°C overnight) with rabbit anti-FLAG antibody (Cell Signaling Technology, Boston, USA), which was used as a primary antibody. The cells were then washed with PBS and incubated with fluorescein isothiocyanate- (FITC-) conjugated fluorescent antibody (Bioss, Beijing, China) for 1 h at room temperature. The nuclei of the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Bioss, Beijing, China) for 5 min and visualized using a confocal laser scanning microscope (Leica, Germany).

DNA fragmentation was visualized by use of the ApopTag kit (Roche, Switzerland). This system labels free 3′OH termini of DNA in cells with digoxigenin-tagged nucleotides with the use of the enzyme terminal deoxynucleotidyl transferase. Total nuclei were stained by 4′,6′-diamidino-2-phenylindole (DAPI, Bioss, Beijing, China). The H9C2 cells were grown on 48-well plates and pretreated with Rapamycin (100 nM) for 2 hours before OGD for 12 hours or 24 hours. Cells only labeled as being TUNEL positive were expressed as percentage of the total nuclei.