Collagen 7

Whole mount cryosections were prepared for immunofluorescence microscopy as previously described (Ridky et al., 2010 (link)). In short, slides were fixed in 4% paraformaldehyde or −20°C methanol, permeabilized as required and blocked with 10% horse serum/PBS, followed by incubation with primary antibodies and secondary antibodies conjugated to fluorophores. Slides were mounted with Prolong Gold Antifade Reagent with DAPI (Life Technologies, Grand Island, NY). The primary antibodies used in this study were collagen-VII (Millipore, Burlington, MA), ß1-integrin (Abcam, Cambridge, MA), ki67, desmoglein-3 (Thermo Fisher Scientific, Carlsbad, CA), HA, keratin-10, keratin-5, loricrin, and filaggrin (Covance, Dedham, MA). To quantify differentiation, the ratio of the area in pixels of keratin-10 positive epidermis to area in pixels of total epidermis was measured as a percentage in ImageJ. This analysis was based previously reported methods (Billings et al., 2015, Natale et al., 2018). Results are the mean of at least 3 technical replicates across at least three biologic replicates from individual donors (± s.d.). Significance was assessed by Welch’s t-test across the biological replicates.

Whole mount cryosections were prepared for immunofluorescence microscopy as previously described (Ridky et al., 2010 (link)). Briefly, slides were fixed in 4% paraformaldehyde or −20°C methanol, permeabilized as required and blocked with 5% goat serum/PBS, followed by incubation with primary antibodies, and secondary antibodies conjugated to fluorophores. Slides were mounted with Prolong Gold Antifade Reagent with DAPI (Life Technologies, Grand Island, NY). The primary antibodies used in this study were IQGAP1 and Collagen VII (Millipore, Billerica, MA), IQGAP3 (Abcam, Cambridge, MA), Desmogelin-3 (Invitrogen, Carlsbad, CA), and Keratin-5 (Covance, Conshohocken, PA).

Whole mount cryosections were prepared for immunofluorescence microscopy as previously described (Ridky et al., 2010 (link)). In short, slides were fixed in 4% paraformaldehyde or −20°C methanol, permeabilized as required and blocked with 10% horse serum/PBS, followed by incubation with primary antibodies and secondary antibodies conjugated to fluorophores. Slides were mounted with Prolong Gold Antifade Reagent with DAPI (Life Technologies, Grand Island, NY). The primary antibodies used in this study were keratin-5, keratin-10, loricrin, and filaggrin (Covance, Conshohocken, PA), Collagen VII (Millipore, Billerica, MA), involucrin (Sigma, St. Louis, MO), phospho-S6 (235/236), phospho-S6 (240/244), cleaved caspase-3, cleaved caspase-8, LC3A (Cell Signaling, Danvers, MA), p-PKC∂1 (Cell Signaling, Danvers, MA), TFAM (gift from C. Cameron, Penn State University), cytochrome c (BD Pharmingen, San Diego, CA), TRF2 (R & D Systems, Minneapolis, MN) and ki67 (ThermoScientific, Fremont, CA).