Immunohistochemical Analysis of Wound Sections

PFA-fixed paraffin sections of human and mouse wounds were dewaxed and rehydrated using a xylene/ethanol gradient followed by antigen retrieval using citrate buffer (10 mM citric acid, pH 6.0) at 95°C for 1 hour. Unspecific binding sites were blocked with 12% BSA in PBS for 1 hour at room temperature. Incubation with primary antibodies (table S4) at 4°C O/N was followed by incubation with biotin-conjugated secondary antibodies (table S5). After each antibody incubation step, extensive washing steps in 0.1% Tween in PBS were performed (3 × 10 min). The VECTASTAIN ABC and DAB peroxidase substrate kits (#PK-6100 and #SK-4100; Vector Laboratories, Burlingame, CA) were used for signal visualization according to the manufacturer’s instructions. Sections were counterstained with hematoxylin and eosin (65 (link)) and mounted with Eukitt. Immunohistochemistry sections were imaged using a 3DHistech Pannoramic 250 slide scanner (3DHistech, Budapest, Hungary).