Hsp47

Adherent cells on passage 7 were resuspended in FACS (fluorescence-activated cell sorting) buffer, and the concentration adjusted to 10⁵cells/mL. For intracytoplasmic and nuclear markers, cells were permeabilized with 5 μl 0.1 % Triton X-100 for 30 min prior to incubation with primary antibodies (concentration of 1:100) specific for stem cells, inflammation, and cell cycle progression: Oct 3/4 (C-10, SC-5279), Nanog (n-17, SC-30331), CD45/OX1 (SC-53045), CD105 (2Q1707, SC-71042), CD90 (Thy-1, aTHy-1A1), CD34 (BI-3C5, SC: 19621), Caspase-3 (SC-7272), HSP-47 (SC-8352), P21 (SC-6246), Ki67 (Ab – 15580), Cyclin-D1 (AB-27618), P53 (Ab −26), TRA-1-81 (SC-21706), MCP-1 (SC- 32771), TNF-R1 (SC, 52746), all from Santa Cruz Biotechnology, as well as CD117 (c-Kit, SCF-Receptor Ab-6, RB-1518-R7, Thermo Scientific, Lab Vision Corporation, Fremont, CA, USA), VEGF-R1 (Clone VG1, M7273, DakoCytomation, CA, USA), COX-2 (Cayman Chemical Co, EUA), CD11b (MCA-551FT), Ly6a (Ab – 51317), CD1a (SC-18885), and CD133 (Mab4310, Merck Millipore), all for 45 min at room temperature. Then, cells were incubated for 2 h with a secondary antibody (Anti-Mouse FITC, DakoCytomation, Santa Cruz Biotechnology). The analysis was performed using a flow cytometer (FACSCalibur, BD). The expression of surface markers was determined by comparison with an isotype control analyzed by a histogram on the CELLQUEST program.

Immunohistology was performed as described previously (Quan et al., 2010 (link)). Briefly, skin samples embedded in OCT were sectioned (7 µm), fixed in 2% paraformaldehyde, permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS), blocked with corresponding serum (5% in PBS), and incubated for one hour at room temperature with SDF-1 (R&D Research, Minneapolis, MN, USA), HSP47, langerin, CD31, CXCR4, and α-smooth muscle actin primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with corresponding secondary antibodies for one hour at room temperature. Between steps, the slides were rinsed for 10 min in Tris buffered saline with 0.1% Triton-X-100 (TBST). All sections were lightly counterstained with haematoxylin. The slides were examined using a digital imaging microscope (Zeiss, Germany). Specificity of staining was determined by substituting corresponding isotype-control immunoglobulins for the primary antibodies.

Immunoblot analysis was performed as previously described [53 (link), 54 (link)] with use of the following antibodies: TGF-β1, collagen I, PDGFR-β, α-SMA, Smad3, p-Smad3 (Cell Signaling Technologies), Fstl1 (R&D Systems), IL-6, HSP47 (Santa Cruz Biotechnology), FSP1 (Sigma-Aldrich), FN1 (Sigma-Aldrich), and HRP-labeled goat anti-mouse IgG or goat anti-rabbit IgG antibody peroxidase-conjugated (ZSGB-BIO, Beijing, China). GAPDH (Hangzhou Goodhere Biotechnology, Hangzhou, China) was used as an internal control for normalization of protein expression.