Goat serum albumin

Myocardial tissues were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin followed by dehydration in graded ethanol solutions and in toluene. Five-micron-thick sections were stained with hematoxylin and eosin (H.E.) or Masson's stain and examined via light microscopy for histopathological analysis. Microvascular density was observed using a CD31 antibody (1:1500, Abcam), eNOS phosphorylation (Ser¹¹⁷⁷) (1:1000, Abacm) and endothelial barrier integrity was assessed with VE-cadherin (1:1000, Abcam). For immunofluorescence staining in vitro, cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 10% goat serum albumin (Invitrogen, USA.). Specimens were subsequently incubated with primary antibodies against Drp1 (1:1000, Abcam) and F-actin (1:1000, Abcam). DAPI staining (Sigma-Aldrich, USA) and mitochondrion-selective MitoFluor™ staining (Molecular Probes, USA) were performed to label nuclei and mitochondria, respectively [32] .

Cells were fixed in 4% paraformaldehyde at room temperature for 10 min and permeabilized in 0.2% Triton X-100. Subsequently, 10% goat serum albumin (Invitrogen; Thermo Fisher Scientific, Inc.) was used to block the cells at room temperature for 1 h. The cells were incubated in a solution containing diluted anti-p53 (1:100) with or without anti-USP10 (1:100) overnight at 4°C and incubated with Alexa Fluor 488 (green) fluorescent secondary antibodies (1:200) at room temperature for 1 h (cat. no. A11008; Invitrogen; Thermo Fisher Scientific, Inc.). The nuclei were counterstained with DAPI at room temperature for 1 min. Images were captured under an Olympus DX51 fluorescence microscope (Olympus Corporation) (magnification, ×400) or an Olympus FV1200 confocal laser scanning microscope (magnification, ×1,000) (Olympus Corporation).

The cells were washed twice with PBS and permeabilized in 0.1% Triton X-100 overnight at 4°C. Subsequently, 10% goat serum albumin (Invitrogen; Thermo Fisher Scientific, Inc) was used to block the samples for 1 hour at room temperature. The sections were then cryoprotected in a PBS solution supplemented with 0.9-mol/L sucrose overnight at 4°C. After neutralization with NH₄Cl buffer, the sections were permeabilized for 45 minutes with 0.05% saponin/PBS (pH 7.4) and incubated with H₂O₂ (3%) for 10 minutes Subsequently, samples were treated overnight at 4°C with the following primary antibodies: OPA1 (1:1000, Abcam, #ab42364), Yap (1:1000; Cell Signaling Technology, #14074), and cyt-c (1:1000; Abcam; #ab90529). Mitochondrial potential was measured using a JC-1 kit (Beyotime Institute of Biotechnology, China). The length of mitochondria was measured using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD). After HR injury, primary cardiomyocytes were washed 3 times with PBS and then incubated with fresh medium supplemented with 10-mg/mL JC-1. Thirty minutes later, the samples were washed 3 times with PBS to remove the free probe, and then fresh medium was added. The samples were observed through fluorescence microscopy (Olympus BX-61). The red/green fluorescence of JC-1 was analyzed using Image-Pro Plus version 4.5 (Media Cybernetics, Inc).