Ab10278

For immunofluorescence (IF) analysis, the samples were harvested from the skin of the hind limb of interest at a size of 2 × 2 cm. Samples were then fixed with 10% formaldehyde for 12 to 14 days at room temperature, embedded in paraffin and sectioned for H&E and IF staining. The slides were rehydrated, and heat‐induced antigen retrieval was performed using 95°C citric acid (S2369, Dako). Nonspecific binding was blocked with antibody diluent (S3022, Dako) for 30 min. Samples were incubated with the following primary antibodies overnight at 4°C: anti‐LYVE‐1 (1:200, ab10278, Abcam), anti‐collagen I (1:200, ab34710, Abcam), anti‐CD3 (1:200, ab699, Abcam), anti‐CD4 (1:200, ab133616, Abcam), anti‐CD45 (1:200, ab10558, Abcam), and anti‐alpha smooth muscle actin (α‐SMA) (1:200, ab21027, Abcam) and anti‐podoplanin (1:200, 14–5381‐85, Invitrogen). IF staining was performed using the following secondary antibodies: Alexa Fluor® 488‐conjugated donkey anti‐rabbit IgG (1:500, ab150061, Abcam), Alexa Fluor® 488‐conjugated goat anti‐mouse IgG (1:500, ab150117, Abcam), Alexa Fluor® 594‐conjugated goat anti‐mouse IgG (1:500, ab150120, Abcam) and Alexa Fluor® 594‐conjugated goat anti‐rabbit IgG (1:500, ab150080, Abcam). Nuclear staining was performed using DAPI.