Histological and Immunohistochemical Analysis of Wound Angiogenesis

For H&E staining, slides with 5 μm tissue sections were baked at 60°C for 30 min and stained with an Autostainer XL (Leica Microsystems, Wetzlar, Germany) using an established protocol (Chen et al., 2020 (link); Chen et al., 2022 (link)). Immunohistochemistry of CD31 was performed in skin wound sections of db/db mice (6 mice per group) as described previously (Chen et al., 2022 (link)). Briefly, for wound tissue analysis of CD31 immunostaining, formalin-fixed paraffin-embedded sections were probed with a 1:50 dilution of anti-CD31 Ab overnight at 4°C, and then 1 h at room temperature with 1:500 dilution of a secondary antibody conjugated with Alexa-555. Images were obtained with a Zeiss LSM 880 confocal microscope. Vascular density, determined from 6 high-power fields per section and represented as a percentage of vascular tissue area occupied by CD31 relative to the total area of the field of view, was analyzed using Zeiss Zen and ImageJ software as described (Chen et al., 2022 (link)).