Fab3688g

Paraffin- or optimal cutting temperature compound-embedded tissues were sectioned and stained6 (link)7 (link) using the following primary antibodies (all diluted 1:100 unless stated otherwise) for immunofluorescence labelling: Lrig: R&D Systems, FAB3688G; CD26: R&D Systems, AF954; Sca1: R&D Systems, AF1226; PDGFRa: R&D Systems, AF1062; Collagen III: Abcam, ab7778, Collagen11a1: Abcam, ab64883; Elastin: Abcam, ab21610; Caveolin: Cell Signaling Technology, 3267; phospho-Histone H3 (Ser10) antibody: Cell Signalling Technology, 970; Active Caspase-3: RnD Systems, AF835; K14: Covance, PRB-155P, 1:500; GFP: Abcam, ab13970, 1:500; RFP: Rockland, 600-401-379, 1:300. EdU staining was performed with a Click-it EdU imaging kit (Invitrogen) according to the manufacturer's recommendations. Images were acquired with a Nikon A1 Upright Confocal microscope. Images of H&E- and Herovici-stained sections were acquired with a Hamamatsu slide scanner. Representative images of skin from two to three independent experiments with at least three biological replicates per group are shown.

Dermal fibroblasts were isolated as previously described (Jensen et al., 2010 (link); Collins et al., 2011 (link); Rognoni et al., 2018 (link)). The muscle and fat from dissected neonatal or adult back skin were scrapped off from the underside of the skin using a scalpel and the tissue was incubated overnight at 4∘C in a Dispase (Sigma) only solution. The epidermis was then carefully peeled off and discarded, leaving the intact dermis. The dermis was minced using a scalpel and enzymatically dissociated with a mixture of 1.25 mg/ml collagenase type 1 (Invitrogen), 0.5 mg/ml collagenase type 2 Worthington), 0.5 mg/ml collagenase type 1V (Sigma), 0.1 mg/ml hyaluronidase IVS (Sigma) and 50 U/ml DNase 1 for approximately 45 min at 37∘C. Dermal cell suspensions were passed through a 70 μm cell strainer and washed three times with PBS before being labeled with the following antibodies: Cd24 PerCP-Cy5.5 (eBioscience, Clone M1/69), Cd26 PerCP-Cy5.5 (eBioscience, Clone M194-112), Ly-6A/E APC (eBioscience, Clone D7), Cd140b APC (Thermo Fisher Scientific, 17-1402-82), Pref-1/Dlk1 APC (R&D Systems, FAB8634A), Lrig1 Alexa Fluor 488-conjugated (R&D Systems, FAB3688G). DAPI was used to exclude dead cells. FACS analysis was carried out with BD FACSCanto 1 or 11. Data analysis was performed using FlowJo software version 10.5.3.