Inverted optical light microscope

Rat cardiac tissues were fixed in 4% paraformaldehyde (Beyotime Institute of Biotechnology) at 4°C for 24 h, embedded in paraffin and cut into 4 µm-thick sections. Following blocking in Immunol Staining Blocking Buffer at room temperature for 15 min, sections were incubated with an anti-von Willebrand Factor (vWF) primary antibody (cat. no. ab6994; 1:200; Abcam) overnight at 4°C. Subsequently, sections were incubated with a biotinylated mouse anti-rabbit IgG secondary antibody (cat. no. bs-0295M; 1:100; BIOSS) at room temperature for 30 min. Sections were stained with dyaminobenzidine (Beyotime Institute of Biotechnology) for 10 min and haematoxylin for 3–5 min at room temperature. Stained sections were visualized and imaged at ×200 magnification using an inverted optical light microscope (Olympus Corporation).

CMVECs (5×10⁴) were seeded into the upper chambers of the Transwell inserts (pore size, 8 µm; EMD Millipore) with FBS-free medium. DMEM supplemented with 20% FBS was plated into the lower chambers. Following treatment with H/R and cell culture for 48 h, migratory cells were fixed in 4% paraformaldehyde at room temperature for 30 min and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) at room temperature for 15 min. Following washing twice with PBS, migratory cells were observed and imaged at ×200 magnification using an inverted optical light microscope (Olympus Corporation) and the number of cells was quantified for analysis.

Rat cardiac tissues were fixed in 4% paraformaldehyde (Beyotime Institute of Biotechnology) at 4°C for 24 h, embedded in paraffin and cut into 4 µm-thick sections. For haematoxylin and eosin (H&E) staining, sections were stained with haematoxylin for 3–5 min and 1% eosin for 5 min at room temperature. For Masson and Sirius red staining, sections were stained using the Masson's Trichrome Stain kit and the Sirius Red Stain kit (both Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocol, respectively. Stained sections were viewed and imaged at ×200 magnification using an inverted optical light microscope (Olympus Corporation).