Stem cell factor (scf)

Cultures were fixed with 4% paraformaldehyde in PBS for 15 min at about 25°C, and then the cells were sealed with 5% bovine serum albumin in PBS for 30 min, followed by incubation with primary antibodies, including nestin (1:200; Sigma-Aldrich, St. Louis, MO, USA), NCAM (1:500; Sigma-Aldrich), and SCF (1:200; Abcam), overnight at 4°C. After washing with PBS, cultures were incubated with FITC-conjugated goat anti-mouse (1:200; ZSGBBIO) at about 25°C in the dark for 2 h. Fluorescence signals were detected with an Olympus fluorescence microscope (Olympus).

Human skin samples were obtained from the Glasgow Caledonian University Skin Research Tissue Bank, Glasgow UK. The tissue bank has NHS research ethics approval to supply human skin for research (REC REF: 16/ES/0069). All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the NHS East of Scotland Research Ethics Service. Informed consent was obtained from all subjects (no patients were under 18 years of age). Samples were formalin fixed and embedded in paraffin following standard procedures prior to sectioning. The sections were then deparaffinized in xylene and treated for 3 h with antigen retrieval solution (Dako) at 80 °C. The sections were cooled to room temperature and blocked with 20% fetal bovine serum for 45 min and then immunostained overnight with 1:100 dilution of primary antibody to PECAM-1 (Cell Signaling), SCF (Abcam), FSP1 (Abcam), c-Kit (Abcam), and mast cell tryptase (Abcam). Secondary staining was performed with secondary antibodies conjugates with Alexa Fluor 488 or 594 dye (Thermo Scientific). Following staining, the samples were imaged using confocal microscopy. For quantification, double-positive areas were quantified using Photoshop and ImageJ.