Anti cd31 clone jc70a

All specimens were stained with hematoxylin and eosin (H&E), Elastica van Gieson, von Kossa, and Masson‐Trichrome (CoverStainer for H&E and ArtisanLink for other stains; Dako). In addition, immunohistochemical staining was performed using the following antibodies: anti‐CD68 (Clone KP1; Dako), anti‐CD10 (Clone 56C6; Novocastra), anti‐CD31 (Clone JC70A; Dako), and anti‐α‐smooth muscle actin (Clone 1A4; Dako). Stained slides were scanned using Aperio Scan Scope and imaged with Aperio Image Scope (Leica Biosystems). H&E‐stained slides were prepared to obtain a general overview over the tissue and to evaluate the thickness of the epidermis and dermis and re‐epithelialization of the wound bed. Von Kossa‐stained slides were used to evaluate the presence of calcification. Elastica van Gieson‐stained slides were used to evaluate the presence of elastic fibres and collagen. Masson trichrome staining was performed for the histological visualisation of collagen and identification of collagen subtypes. The amount of mature collagen within the wound area was assessed in a semi‐quantitatively manner by scoring for a substantial amount of collagen, a moderate amount of collagen, a low amount of collagen, or no collagen. The biopsies were analysed in a paired fashion: PCT‐treated wounds were compared to SoC‐treated wounds at V9 and to initial skin biopsies taken at V2 (Figure S2).

Formalin-fixed 4 μm sections were deparaffinised with xylene and rehydrated through graded ethanol and demineralised water. Antigens were retrieved by boiling in 10 mM citrate buffer, pH 6.0 in a pressure cooker. Non-specific binding was blocked using Antibody Diluent S2022 (Dako). Consecutive tissue sections were incubated with the following primary monoclonal mouse anti-human antibodies diluted in Antibody Diluent S2022 (Dako): anti-TF (No. 4509, American Diagnostica), anti-Epithelial Membrane Antigen (MUC1) (clone E29), anti-CD68 (clone KP1) and anti-CD31 (Clone JC70A) from Dako. After blocking endogenous peroxidases, staining was visualised using a peroxidase-labelled polymer conjugated to goat anti-mouse immunoglobulins and 3,3′-diaminobenzidine (K4007, Mouse EnVision⁺ kit, Dako). Sections were decolourised, counterstained with haematoxylin (1 : 1 dilution with demineralised water; Merck, Darmstadt, Germany), decolourised again, dehydrated and finally permanently mounted.
Mouse IgG1 (BD Biosciences) and omission of the primary antibody served as specificity controls. Normal pancreatic islet cells, tonsils and ductal breast cancer cells served as positive control for TF, CD68 and MUC1 staining, respectively. No separate control was prepared for CD31 staining.