Laminin

Total protein of exosomes, cells, or spinal cord tissues was extracted using RIPA buffer and protein concentration was evaluated with a BCA assay according to the manufacturer’s protocol. Perform WB experiments according to the conventional process; all primary antibodies included CD9 (rabbit), CD63 (rabbit), GAPDH (rabbit), β-actin (rabbit), CTGF (rabbit), GFAP (rabbit), vimentin (rabbit), fibronectin (rabbit), laminin (rabbit), BDNF (rabbit), and TGF-β1(rabbit, Abcam, UK).

Adult porcine esophagi were donated from Mary’s Ranch, a USDA-certified slaughterhouse (Pembroke Pines, FL, USA). The decellularizing agents Triton X-100, ammonium hydroxide, trypsin, EDTA, DNase I, RNase, phosphate buffered saline (PBS), sodium deoxycholate (SD), ethylenediaminetetraacetic acid (EDTA), and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific (Waltham, MA, USA). An antibiotic/antimycotic (AA) containing penicillin, streptomycin and amphotericin-B was purchased from Gibco (Carlsbad, CA, USA). A human esophageal squamous cell carcinoma cell line (KYSE30) was purchased from Sigma-Aldrich (St. Louis, MO, USA). A human primary esophageal fibroblast cells and related culture medium were obtained from Cell Biologics Inc (Chicago, IL, USA). RPMI 1640 cell culture medium was purchased from Gibco (Carlsbad, CA, USA). Primary antibodies collagen type 1, fibronectin, laminin, and periostin were purchased from Abcam (Cambridge, MA, USA). Immunohistochemistry reagents were purchased from Vector Laboratories (Burlingame, CA, USA). The Quant-iT Pico-Green dsDNA Assay Kit was purchased from Thermo Fisher Science (Waltham, MA, USA).

Western blot analysis was carried out with standard procedures and followed primary antibodies against Laminin (1:2000, Abcam, Cambridge, UK), HGF (1:2000, Abcam, Cambridge, UK), and Cx43 (1:1000, Sigma, St. Louis, MO, USA). IRDyeTM 800-conjugated goat anti-rabbit IgG (1:10,000, LI-COR Biosciences, Lincoln, NE, USA) was applied as secondary antibodies. Protein expression was visualized and quantified by the LI-COR Odyssey^® Imaging System and Odyssey^® software (LI-COR Biosciences, Lincoln, NE, USA), respectively. Results were normalized relative to β-Tubulin (1:1000; Cell Signaling, Beverly, MA, USA) expression to correct for variations in protein loading and transfer.

Lung tissues were harvested and fixed in 4% paraformaldehyde overnight. The tissues were subsequently washed in PBS and dehydrated in in 30% sucrose overnight. The tissues were embedded in optimal-cutting-temperature (OCT) compound (Sakura Finetek) and then frozen at −80°C. Tissue blocks were cut into 20-μm-thick sections and permeabilized in cold acetone. The sections were blocked with serum-free protein block (Dako) and immunolabeled with antibodies against mouse CD3 (2A7; BioLegend), Gr-1 (RB6-8C5; BD Biosciences), CD169 (3D6-1.12; AbD Serotec), and laminin (rabbit polyclonal; Abcam). The slides were mounted with ProLong Gold antifade agent (Thermo Fisher). Images were acquired using a confocal microscope (Olympus FV3000) using a 10× and 20× (0.95-numerical-aperture [NA]) objective and processed using Imaris (v9.5; Bitplane).

The tissues of the wound site were retrieved 12 days after the treatment and embedded in an optimal cutting temperature compound (TISSUE-TEK^® 4583, Sakura Finetek USA Inc., Torrance, CA, USA), followed by freezing and slicing into 10-μm thick sections at −22 °C. Immunohistochemical analysis of laminin (Abcam, Cambridge, UK) and involucrin (Abcam) was performed to examine the regeneration of the basal layer and epidermis, respectively. The staining signals for laminin and involucrin were visualized using fluorescein isothiocyanate-conjugated or rhodamine-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA), respectively. To assess the microvessel density, tissue slices were stained with antibodies against vWF (Abcam). vWF-positive signals were visualized using fluorescein isothiocyanate-conjugated secondary antibodies (Jackson Immuno Research Laboratories). The tissue slices were mounted in 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories). A fluorescent microscope (Olympus) was used to count the microvessels. Four different images per slide from 20 random slides were randomly analyzed for each group (four different samples per group) for immunohistochemical quantification.

Cells were fixed in 4% paraformaldehyde in PBS for 10 min and myobundles were fixed in 2% paraformaldehyde in PBS overnight at 4°C. Following fixation, samples were washed in PBS then blocked in 5% chick serum with 0.2% Triton-X 100. The following primary antibodies were used for tissue characterization: desmin (SCBT, Dallas, TX, 1:200), anti-GFP (Life Technologies, 1:200), laminin (Abcam, Cambridge, MA, 1:200), muscle creatine kinase (SCBT, 1:100), MyoD (BD, 1:100), myogenin (SCBT, 1:100), myosin heavy chain 1/2/4/6 (SCBT, 1:100), Pax7 (DSHB, Iowa City, IA, 1:50), sarcomeric α-actinin (Sigma, 1:200), and vimentin (Sigma, 1:200). Corresponding fluorescently labeled secondary antibodies (1:200), α-bungarotoxin (1:100), and phalloidin (1:200) were purchased from Life Technologies. Oil Red O staining was performed using standard protocols on cryosections of myobundles fixed in 4% paraformaldehyde. Hematoxylin and eosin stain was performed on paraffin embedded sections of 2% paraformaldehyde fixed myobundles using Harris modified hematoxylin (Sigma) and Eosin Y (Sigma). Images were acquired using a Zeiss 510 inverted confocal microscope and analyzed using LSM Image Software. Mosaic images for fiber length measurements were generated using Mosaic J in FIJI.