Filaggrin Proteins

To evaluate epidermal thickening, the ear and dorsal skin of each mouse was obtained on day 21 and fixed in 10% neutral buffered formalin. Tissues were embedded in paraffin and sliced into 5-μm-thick sections. Sections were then transferred to probe-on-plus slides (Thermo Scientific, Carlsbad, CA, USA), and deparaffinized skin sections were stained with H&E. Mast cells in the skin were stained with toluidine blue. Some sections were stained for immunohistochemical markers using anti-filaggrin (1:1000; ab24584; Abcam), anti-involucrin (1:1000; ab68; Abcam), anti-loricrin (1:1000, ab85679, Abcam), anti-occludin (1:1000; 33-1500; Invitrogen), anti-PAR-2 (1:100; sc-8205, Santa Cruz Biotechnology), and anti-TSLP (1:500; NB110-55234; Novus Biologicals) antibodies. The staining procedure was performed with an Ultravision Quanto Detection System (TL-060-QHD; Thermo Scientific). The slices were washed with PBS, dehydrated and mounted in Permount (SP15-100, Thermo Scientific). All stained sections were then examined by light microscopy (DM750, Leica, Wetzlar, Germany) to assess histological changes. Morphometric analysis (immunostained area of the epidermis to each analyzed protein in relation to total area of the epidermis) was performed using ImageJ 1.51 software (National Institutes of Health, Bethesda, MD, USA). All histological examinations were analyzed in 3 sections/animal slices.

The fixed slices were treated with 3% H₂O₂ and then blocked with 5% BSA. The specimens were finally incubated with anti-Filaggrin (Santa Cruz, California, United States) or anti-Involucrin (1: 500; Absin, Shanghai, China) antibody overnight at 4°C, and then incubated with horseradish peroxidase-conjugated secondary antibody at 37°C for 4 h followed by reaction with 3,3-diaminobenzidine (DAB).

The presence of ECM and BM-related proteins was assessed with fluorescent confocal microscopy. The constructs and grafts were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4°C, then transferred to a 30% sucrose solution for 24 hours at 4°C, and lastly embedded in optimal cutting temperature (O.C.T.) compound for cryosectioning (section thickness = 16 μm). For immunofluorescent staining, the slides were dried overnight at room temperature in the dark, then permeabilized with 0.1% Triton X, blocked with 8% bovine serum albumin and 5% donkey serum in PBS for 1 hour, and incubated overnight at 4°C with the following antibodies: keratin 14 (BioLegend, #906004), involucrin (Abcam, #ab27495), human-specific involucrin (Abcam, #ab68), loricrin (Abcam, #ab198994), filaggrin (Abcam, #ab221155), desmoglein 1 (Progen, #651111), desmoglein 3 (Invitrogen, #326300), collagen type I (Abcam, #ab6308), collagen type III (Abcam, #ab6310), collagen type IV (Abcam, #ab6586), collagen type VII (Abcam, #ab6312), collagen type XVIII (Santa Cruz Biotechnology, #sc32720), CD90 (BioLegend, #328110), FAP (R&D Systems, #AF3715-SP), smooth muscle actin (Abcam, #ab5694), fibronectin (Santa Cruz Biotechnology, #sc-73611), laminin α5 (EMD Millipore, #MABT39), nidogen (Abcam, #ab254325), elastin (Abcam, #ab21610), Ki67 (Abcam, #ab16667), and cleaved caspase 3 (Cell Signaling Technology, #9664). Different combinations of Alexa Fluor Plus secondary antibodies (Invitrogen) were used to detect the primary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Staining of the F-actin fibers was performed using phalloidin conjugated with Alexa Fluor 594 (Invitrogen, #A12381). Hyaluronic acid was stained using biotinylated versican G1 domain (Amsbio, #AMS.HKD-BC41) and Alexa Fluor 555–conjugated streptavidin (Invitrogen, S21381). To differentiate human and murine vasculature in tissue sections, we simultaneously stained the ECs with an anti-CD31 antibody (Abcam, #ab28364) directed both against human and mouse and with griffonia simplicifolia lectin I isolectin B4, DyLight 594 (Vector Laboratories, #DL-1207-.5), which stains the endothelium of several nonprimate animals including mice. 3D imaging of the vasculature was performed through whole-mount staining following the same protocol described for the tissue sections, with the exception of using 0.1% Triton X in all the staining and washing solutions. Anti-CD31 (Abcam, #ab28364) and anti-vimentin (Santa Cruz Biotechnology, #sc-6260) were used as primary antibodies. The tissue was cleared using Ce3D Tissue Clearing solution (BioLegend, #427704) for 20 min on a rocking platform before proceeding with the imaging. TUNEL assay for the detection of DNA fragmentation was performed on frozen sections of the grafts using Click-iT TUNEL Alexa Fluor 647 Imaging Assays for Microscopy & HCS (Thermo Fisher Scientific, #C10247) following the manufacturer’s recommendation. The images were acquired using a Leica Stellaris 5 (Leica, Germany). Three biological replicates were analyzed for each time point and sample.