For HE and Masson staining, 4-μm-thick paraffin-embedded sample slices were prepared. These experiments were performed by Servicebio (CHN). Images were taken using an Olympus1X71 microscope (Olympus, JPN). Image-Pro Plus 6 (Media Cybernetics, USA) was used to measure the thickness of the epidermis and evaluate the integrity of the new skin in the HE staining. Collagen was quantified by calculating the ratio of the positive area (blue–green collagen) to the total area in the skin tissue Masson staining.
In order to detect the presence of CD31, immunohistochemistry was carried out. Rehydrated paraffin sections were incubated in a microwave oven for 8 min. All sections were blocked with 3% BSA at 37 °C for 30 min, followed by incubation with anti-CD31 antibody (Servicebio, GB11063-3, 1:800) at 4 °C overnight. After washing with PBS, a goat anti-rabbit secondary antibody (Servicebio, GB23303, 1:200) coupled with horseradish peroxidase was employed. Samples were incubated at 37 °C for 1 h. Sections were visualized with diaminobenzidine tetrahydrochloride, and staining was detected with a light microscope (Olympus, JPN). CD31 levels were quantified by calculating the ratio of the positive area (yellowish-brown) to the total area using Image-Pro Plus 6 (Media Cybernetics, USA).
In order to detect the presence of CD31, immunohistochemistry was carried out. Rehydrated paraffin sections were incubated in a microwave oven for 8 min. All sections were blocked with 3% BSA at 37 °C for 30 min, followed by incubation with anti-CD31 antibody (Servicebio, GB11063-3, 1:800) at 4 °C overnight. After washing with PBS, a goat anti-rabbit secondary antibody (Servicebio, GB23303, 1:200) coupled with horseradish peroxidase was employed. Samples were incubated at 37 °C for 1 h. Sections were visualized with diaminobenzidine tetrahydrochloride, and staining was detected with a light microscope (Olympus, JPN). CD31 levels were quantified by calculating the ratio of the positive area (yellowish-brown) to the total area using Image-Pro Plus 6 (Media Cybernetics, USA).