Anti collagen type 1 antibody

Recombinant human (rh) IL-6, IL-1α, TGF-β1, TNF-α, IL-8 and granulocyte macrophage colony-stimulating factor were purchased from R&D Systems (Abingdon, UK). rhIL-1β and gelatin solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle Medium cell culture media, antibiotics, L-glutamine, sodium pyruvate and trypsin-EDTA were purchased from Invitrogen (Eugene, OR, USA). Fetal calf serum (FCS) was from Lonza (Levallois, France). Acrylamide, sodium dodecyl sulfate (SDS), Tris, and bovine serum albumin were purchased from Eurobio (Les Ulis, France). Anti-collagen type I antibody was purchased from Millipore (Billerica, MA, USA). Anti-STAT3 and p-STAT3 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-IL6R antibody was purchased from Abcam (Cambridge, UK). Anti-HSC70 antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Polyclonal secondary immunoglobulins/HRP were purchased from Dako (Les Ulis, France). The Pierce^™ BCA protein assay kit, LDS sample buffer, MOPS SDS running buffer, 4–12% Bis-Tris Gel, Pierce^® ECL western blotting substrate, antioxidant and sample reducing agent were purchased from Thermo Fisher Scientific (Saint-Herblain, France). Tris/glycine migration buffer and Trans-blot^® Turbo^™ transfer pack were purchased from Bio-Rad (Hercules, CA, USA).

Cells on coverslips were washed thrice with PBS and then were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After washing 3 times with PBS, the samples were permeabilized with 0.5% Triton X-100 in PBS for 10 min, rinsed with PBS, and then immunostained with anti-collagen type I antibody (Millipore; Billerica, MA, USA) overnight at 4°C. After washing 3 times with PBS, the coverslips were exposed to Alexa Fluor 594-labeled secondary antibodies (Molecular Probes, Inc., Eugene, OR, USA) for 1 h; then, the nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Co., St. Louis, MO, USA). The coverslips were mounted with anti-fade solution (Molecular Probes). Images of samples from independent experiments were captured using a fluorescence microscope. A total of 6 representative images per sample were scanned with a 400 times magnification in a total of 1760 digital images in TIFF file extension format, and the stain intensity of scanned images were analyzed to quantify the total amount of collagen type I using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). For each sample, staining without primary antibody was performed with a side-by-side parallel specimen as a negative control; these stains all yielded blank images.