Anti alpha smooth muscle actin antibody

Explanted grafts were incised in longitudinal direction and fixed in 4% paraformaldehyde for 48 hours. After fixation, explanted grafts were divided into proximal and distal parts and subsequentially embedded in paraffin. In each part, sections with 6 μm thickness were prepared consecutively and subjected to hematoxylin and eosin, Elastica van Gieson (EVG), Sirius red and von Kossa staining respectively. Immunostaining of von Willebrand factor for endothelium (anti-von Willebrand Factor antibody, Abcam, Cambridge, UK, 1:3000), α-smooth muscle actin for smooth muscle cells (anti-alpha smooth muscle actin antibody, Abcam, 1:500), CD45 for leukocytes (rabbit polyclonal anti-CD45 antibody, ab10558, Abcam, 1:10000), CD68 for porcine macrophages (mouse anti pig macrophages antibody, clone BA4D5, Bio-Rad Antibodies, Puchheim, Germany, 1:50) and CD90 (human/porcine/canine CD90/Thy1 antibody, R&D Systems, Minneapolis, MN, 1:20) were performed, respectively. Sirius red staining sections were observed using polarized light microscope (BX51; Olympus, Tokyo, Japan). For the quantification of collagen and elastin after decellularization, Sirius red and EVG staining were used respectively (2 samples for each staining), and quantified by automatic measurement of an all-in-one microscope (BZ-X800, Keyence, Osaka, Japan).

After preparing the femoral sections using the same procedure as above, the sections were immunohistochemically processed using an anti-alpha smooth muscle actin antibody according to the manufacturer’s instructions (Abcam, United Kingdom). Briefly, sections were washed with TBS solution containing 0.025% Triton X-100 and then closed for 2 h at room temperature using TBS solution containing 10% normal serum and 1% BSA. rabbit antisera against mouse alpha-smooth muscle (Abcam, United Kingdom) applied to sections overnight at 4°C. To visualize the antigen-antibody reaction, the slides were incubated in a TBS solution containing 0.3% H2O2 for 15 min. Finally, sections were counterstained with hematoxylin.

Thioacetamide was purchased from Sigma–Aldrich, USA. DAB substrate and DAB staining solution were purchased from BD Pharmingen (San Diego, CA 92121, USA). The HepG2 and FL83B cell lines were purchased from ATCC (Manassas, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Thermo Scientific, USA. L-Glutamine (200 mM) was purchased from Sigma–Aldrich, USA. A hydroxyproline assay kit was purchased from Chondrex (WA, USA). Anti-alpha smooth muscle actin antibody-Lot:GR283004-24 (Abcam, Cambridge, UK), goat anti-rabbit IgG H&L (Alexa Fluor1 488) Lot:GR306624-1 (Abcam, Cambridge, UK), hepatitis B core antigen Lot:#SF2406841H (Invitrogen, Thermo Fisher Scientific, South Korea), and goat anti-rabbit IgG (HRP) Lot:GR247075-7 (Abcam, Cambridge, UK) were used. A plasmid DNA purification kit was purchased from Intron Biotechnology (Lynnwood WA). The DNA isolation kit was purchased from QIAamp (DNA Mini kit, Germany). An ELISA kit for HBV antigen analysis was purchased from Wanti-Biopharm (Beijing). All other reagents used were of analytical or chromatographic grade.

Dulbecco's Modified Eagle Medium (DMEM) was from Gibco (Life Technologies, Carlsbad, CA). Primary antibodies used include anti-cleaved caspase-8, anti-cleaved caspase-9, anti-cleaved caspase-3, anti-cleaved PARP, anti-β-actin (Cell Signaling Technologies, Danvers, MA), anti-Fas Ligand, anti-alpha smooth muscle Actin antibody (Abcam, Cambridge, MA), anti-CD68 (AbD Serotec, Raleigh, NC), Rat Anti-Mouse Fas Ligand Monoclonal Antibody for neutralization, Rat IgG1 Isotype Control (R&D Systems, Minneapolis, MN), Anti-Mouse Fas Ligand FITC, Armenian Hamster IgG Isotype Control FITC (eBioscience, San Diego, CA). Fluorophore-conjugated secondary antibodies and 4′6-diamidino-2-phenyl-indole, dihydrochloride (DAPI) were purchased from Molecular Probes (Life Technologies, Carlsbad, CA). Horseradish Peroxidase (HRP)-conjugated Antibodies were purchased from Bio-Rad (Hercules, CA). In Situ Cell Death Detection Kit was from Roche Applied Science (Indianapolis, IN). siRNAs used in this study were Silencer Select siRNAs from Ambion (Life Technologies, Carlsbad, CA). Other chemicals and reagents if not specified were purchased from Sigma-Aldrich (St. Louis, MO).