Acetone

Left ventri cular myocardial tissue samples were cut into slices and fixed with 2.5% glutaraldehyde (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), post-fixed with 1% osmium tetraoxide (Absin Bioscience Inc., Shanghai, China), rinsed with phosphoric acid rinse solution (Beyotime Institute of Biotechnology), dehydrated using a series of acetone (Beyotime Institute of Biotechnology) at different concentrations, embedded and solidified, and cut into slices (thickness, 50-100 nm). The ultrathin slices were stained using 3% uranyl acetate (Shanghai Fortune Biological Technology Co., Ltd., Shanghai, China) and lead nitrate (Tanyun Industry Fine Chemical Co., Ltd., Yingkou, China) at 37°C for 30 min. Samples were observed under a transmission electron microscopy and the images were saved.

The immunofluorescence experiments were performed as previously described (31 (link)). The excised corneas from the CNV assay were rinsed in PBS and fixed in acetone (Beyotime Institute of Biotechnology, Inc.) for 30 min. Following washing and blocking with 2% BSA in PBS for 2 h, the corneas were stained overnight at 4°C, with a rabbit anti-mouse LYVE-1 antibody (cat. no. ab14917; 1:500 dilution; Abcam) and a rat anti-mouse cluster of differentiation (CD)31 antibody (1:100; cat. no. 550274; BD Pharmingen, San Diego, CA, USA). On day 2, the tissue was washed three times in PBS and stored at 4°C in the dark. LYVE-1 antibody was detected with an Alexa Fluor^® 647-conjugated goat anti-rabbit IgG antibody (1:200; cat. no. A-21244; Invitrogen; Thermo Fisher Scientific, Inc.) and the CD31 antibody was detected with Alexa Fluor 488^®-conjugated goat anti-rat IgG polyclonal antibody (1:200; cat. no. A-11006 Invitrogen; Thermo Fisher Scientific, Inc.).
The stained whole mounts were analyzed with a fluorescence microscope (EVOS f1; Thermo Fisher Scientific, Inc.). Each whole mount picture was quantified using Image J software version 1.24o (National Institutes of Health, Bethesda, MD, USA) analysis software as described previously (32 (link)). The mean vascularized area of the suture placement was defined as being 100%; vascularized areas were then relative to this value (vessel ratio).