Immunofluorescent Analysis of Extracellular Matrix and Basement Membrane Proteins

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.