Sca 1

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.

For histology, neonatal (p5) and adult (p112) mice caudal level (c) 4/5 was injured and c5/6 was used as an internal control with 3 animals per group. For fluorescence imaging and immunofluorescence of sections, tail segments were fixed in 4% paraformaldehyde and frozen in OCT medium. Alternating sagittal cryosections (12 μm) were collected to capture the IVD from outer AF to the opposite outer AF. Immunostaining was carried out using antibodies against Sca-1 (R&D Systems), type I collagen (Abcam), nucleostemin (Neuromics), F4/80 (Affymetrix), α-SMA (Sigma), PECAM (CD31) (BD Pharmingen), MCAM (CD146) (Santa Cruz), and CK19 (Abcam), with DAPI counterstaining (1:1000) to visualize cell nuclei. Primary and secondary dilutions used are listed in Supplemental Table 2. EdU and TUNEL assays were performed using the Click IT EdU and Click IT TUNEL kits (Life Technologies), according to manufacturer’s instructions with appropriate positive controls (Supplemental Fig. 4). All images were acquired using Zeiss Axio Imager microscope; an Apotome was used for optical sectioning of fluorescent images.