3.10. Evaluation of Angiogenesis through CD31 Immunohistochemical Staining

After three weeks post-surgery, the mice were euthanized, and the left ventricle was harvested, rinsed with PBS, and fixed in a 4% paraformaldehyde solution for 24 h. The tissue was then rinsed with running water for 1 h and dehydrated using a gradient of an ethanol solution. After dehydration, the tissues were subjected to clearing by immersion in a mixed solution of xylene and anhydrous ethanol for 1 h, followed by immersion in xylene for 1 h. Subsequently, the cleared tissues were embedded in liquid paraffin and sectioned into 5–10 slices of 5 μm thickness. The sections were deparaffinized in xylene for 10 min and rehydrated using a gradient of ethanol solutions, followed by a 5 min wash in running water. The tissue sections were then immersed in an EDTA antigen retrieval buffer, washed three times with PBS, and circled with an immunohistochemistry pen. Within the circles, a 5% BSA solution was added and incubated for 30 min for blocking. After drying, the tissue sections were incubated overnight at 4 °C with a CD31 primary antibody. Following PBS washing and drying, a secondary antibody was added within the circles and incubated for 1 h. After PBS washing, the tissue sections were dried and incubated with DAB for color development, with careful monitoring under a microscope. The staining was terminated by immersion in water once the desired color was achieved. After dehydration in ethanol, clearing in xylene, and mounting with neutral resin, the tissue sections were observed under an optical microscope. Images were captured at 200× and 400× magnification, and the number of CD31-positive capillaries per unit area (mm²) of myocardial tissue was calculated using the ImageJ 1.5.4 software.