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).
Type 1 collagen
Manufactured by Abcam
Sourced in United States, United Kingdom
Type I collagen is a fibrillar collagen that is a major structural component of skin, bone, tendon, ligament, and other connective tissues. It is the most abundant collagen in the human body.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Abcam (10 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
α sma, by Merck Group (5 mentions)
Type 3 collagen, by Abcam (5 mentions)
α sma, by Abcam (5 mentions)
Vimentin, by Abcam (4 mentions)
Type 2 collagen, by Abcam (4 mentions)
Fibronectin, by Santa Cruz Biotechnology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
P smad3, by Cell Signaling Technology (3 mentions)
Fibronectin, by BD (3 mentions)
β catenin, by Abcam (3 mentions)
Gapdh, by Abcam (3 mentions)
P stat3, by Cell Signaling Technology (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Sm22α, by Abcam (2 mentions)
Pdgfrβ, by Cell Signaling Technology (2 mentions)
Pdgfrα, by Cell Signaling Technology (2 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Scgb1a1, by Santa Cruz Biotechnology (2 mentions)
57 protocols using type 1 collagen
1
Decellularization of Porcine Esophagi
2
Immunocytochemical Analysis of Protein Expression
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To evaluate protein expression and localization, immunocytochemical staining was performed. Before using the cell culture slides, they were coated with poly-L-lysine for 4 h, after which they were exposed to UV light for 6 h. Cells were seeded on the poly-L-lysine coated slides. Subsequently, the cells were fixed with 4% paraformaldehyde and permeabilized with phosphate-buffered saline containing 0.01% Triton X-100 for 10 min. Next, the cells were blocked with 3% bovine serum albumin for 1 h, incubated with primary antibodies; α-SMA (Millipore), Fibronectin (Santa Cruz Biotechnology, Inc), and collagen type 1 (Abcam), followed by incubation with secondary antibodies (Invitrogen). Finally, the cells were counterstained with 4’-6-diamidino-2-phenylindole for 10 min and the stained cells were visualized using a confocal laser scanning microscope (LSM700, Zeiss, Oberkochen, Germany).
3
Histological Analysis of Aortic Valve Cusps
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In a representative subset of aortic valve cusp specimen, histological analyses were performed. Aortic valves of patients without (n = 9) or with LVAD (n = 11) were snap‐frozen in cryo compound, and 5 μm sections were prepared. Sections were stained according to standard protocols for haematoxylin/eosin, Movat's pentachrome and von Kossa staining as previously described.14 Immunohistochemical staining for detection of α‐SMA (Sigma) and vimentin as well as staining for CD3, CD86, elastin, collagen type 1 (Abcam), and biglycan (Santa Cruz) were performed as previously described.15 Analyses and documentation were performed in a blinded fashion using pseudonyms for sample labelling with a Leica DM2000 and Leica LAS software Version 3.8.0.
4
Immunohistochemical Analysis of Mouse Lungs
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Mouse lungs were inflated and fixed in 2% paraformaldehyde, dehydrated in a series of increasing ethanol concentration washes, embedded in paraffin and sectioned. Antibodies used were anti-SM22α (goat anti-SM22α 1:200 Abcam), GFP (goat anti-GFP 1:100 Abcam, rabbit anti-GFP 1:100 Molecular Probe), Scgb1a1 (goat anti-Scgb1a1 1:20 Santa Cruz), SPC (rabbit-anti SPC 1:500 Chemicon), Pdgfrα (rabbit anti-Pdgfrα 1:50 Cell Signaling), Pdgfrβ (rabbit anti-Pdgfrβ 1:100 Cell Signaling), vimentin (rabbit anti-vimentin 1:100 Santa Crux), collagen type1 (rabbit anti-Col1 1:500 Abcam), Ki67 (rabbit anti-Ki67 1:50 Abcam), PCNA (mouse anti-PCNA 1:50 Biocare), PO4-Histone H3 (mouse anti-PO4-Histone H3 1:200 Cell Signaling), TubbIV (mouse anti-TubbIV 1:20 Biogenex), S100A4 (rabbit anti-S100A4 1:200 Abcam). LacZ staining of lungs was performed as previously described8 (link). The slide was imaged on a Zeiss LSM 710 confocal microscope and analyzed in ImageJ software.
5
Immunohistochemical Analysis of Skin Regeneration
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IHC was carried out using specific markers, cytokeratin 14 and collagen type 1 (Abcam®, MA, United States) to evaluate, respectively, the regenerated skin’s maturity for keratinocytes and the fibroblasts maturity. The hydrogels were then sectioned with a cryostat and mounted on glass slides. The fixed tissue sections were then blocked with 10% goat serum (Sigma Aldrich®, MO, United States) to prevent non-specific binding of the primary antibody. The tissue sections were incubated with a primary antibody that specifically binds to the protein of interest. The tissue sections are then incubated with a secondary antibody that recognizes the primary antibody Alexa 488 (Abcam®, MA, United States). The labelled secondary antibody is detected using fluorescence microscopy. Finally, the tissue sections counterstained with DAPI to visualize the nuclei and provide morphological context.
6
Immunohistochemical Analysis of Mouse Lungs
7
Cardiac Fibroblast Protein Extraction and Western Blot Analysis
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Proteins were extracted from cardiac fibroblasts or mouse myocardium by RIPA lysis buffer (Applygen, China) containing protease inhibitor cocktail (Sigma, Santa Clara, CA, USA). Total protein concentrations were measured by BCA assay (Pierce, USA). Proteins were then separated by SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Germany). Membranes were incubated with primary antibodies collagen type I, Fibronectin (FN), Sec61α (Abcam, Cambridge, MA), TGF-β1, a-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) (Santa Cruz Biotechnology, CA, USA), α-Tubulin (Bioworld, USA), or Nogo-C (Abmart, China) and then incubated with secondary antibodies goat anti-rabbit IgG, goat anti-mouse IgG, rabbit anti-goat IgG (Biodragon, China), or mouse anti-rabbit IgG LCS (Abbkine, California, USA). Immunoblots were evaluated using the Chemi Doc XRS + instrument (Bio-RAD, Hercules, CA, USA).
8
Exosomal Protein Characterization from Plasma and CM
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The vesicle pellet obtained by use of the Total exosome isolation (TEI) reagent was lysed in 50 μl of RIPA buffer supplemented with complete protease-inhibitors (Roche Diagnostics, Mannheim, Germany) and PhosSTOP phosphatase-inhibitors (Roche Diagnostics). Lysates were sonicated twice (15 s) in a 4°C water bath. Protein concentrations were quantified using the Micro BCATM Protein assay kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Twenty five microgram of vesicles extracted from plasma or CM were for western blotting. Protein expression was assessed using antibodies against CD63, TSG101 (both 1:200, Santa Cruz Biotechnology, Dallas, TX, USA), β-actin, vimentin (1:5000 and 1:500 respectively, Sigma–Aldrich, St.Louis, MO, USA), collagen type I (1:1000, Abcam, Cambridge, UK), and PDGFRβ (1:1000, GeneTex, Irvine, CA, USA). Calreticulin (1:1000, Cell Signaling Technology, Danvers, MA, USA) was used to verify absence of cell contamination. As positive control, a protein cell lysate of the HepG2 cell line was used.
9
Molecular Mechanisms of Cdh1, Cdc20, and APC in Cell Cycle Regulation
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The SIRT1 and AROS constructs used in this study were previously described32 (link). Full-length Cdh1, Cdc20, APC2, and APC11 were amplified via polymerase chain reaction (PCR) and subcloned into the plasmids Flag, Myc-tagged pcDNA3 (Thermo Fisher Scientific), pEGFP-C3 (BD Biosciences) and pGEX4T-1 (GE Healthcare). The antibodies used included the following: SIRT1 (Santa Cruz Biotechnology, sc15404), AROS (Santa Cruz Biotechnology, sc-86209; Abcam, ab201091), Cdh1 (Abcam, ab3242), Cdc20 (Santa Cruz Biotechnology, sc13162), Cyclin B1 (Santa Cruz Biotechnology, sc245), APC2 (Thermo Fisher Scientific, RB-067), APC11 (Abcam, ab154546), p53 (Santa Cruz Biotechnology, sc-126), p21 (Santa Cruz Biotechnology, sc-6246), p16 (Calbiochem, NA29), green fluorescent protein (GFP; Santa Cruz Biotechnology, sc-8334), polyubiquitinated conjugates (poly-Ub; Enzo Life Science, BML-PW8805-0500), acetylated-lysine (Cell Signaling Technology, 9814), hemagglutinin (HA; Merck Millipore, 05-904), Myc (Merck Millipore, 05-724), Flag M2 (Sigma‒Aldrich, F1804), LSD1 (Abcam, ab17721), H3K9me2 (Abcam, ab1120), H3K9ac (Abcam, ab12179), α-SMA-Cy3 (Sigma, C6198), collagen type I (Abcam, ab34710), elastin (Abcam, ab21600), fibronectin (Abcam, ab2413), and β-actin (Santa Cruz Biotechnology, sc47778).
10
Immunofluorescent Staining of Cultured Cells
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Immunofluorescent staining of the cultured cells was performed according to previously described protocols [21 (link)]. Primary antibodies collagen type I (Abcam, Cambridge, UK), fibronectin (Abcam), N-cadherin (Abcam), and vimentin (Abcam) were used. Images were obtained with a confocal microscope (LSM510; Carl Zeiss).
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