Vimentin m7020

Cells were washed with phosphate buffered saline (PBS) and then lysed on ice in RIPA buffer (Sigma Aldrich) containing Phosphatase inhibitor cocktail 2 (Sigma Aldrich), Complete protease inhibitor cocktail (Roche), 100mM NaF, 4mM Na₃VO₄, 1mM phenylmethylsulfonylfluoride (PMSF), 1mM dithiothreitol (DTT) and 100U/ml benzonase (Sigma Aldrich). Samples were resolved by SDS-PAGE on 4–12% Novex Bis-Tris precast gels (Thermo Fisher Scientific) and analyzed by western blotting using the following antibodies: fibronectin sc-6952 (Santa Cruz Biotechnology), YAP #14074, TAZ #4883, Smad2 #5339, tubulin #2148, phospho-YAP (Ser127) #13008 (Cell Signaling Technologies), αSMA ab5694, GAPDH ab9485, Smad3 ab40854, phopsho-Smad3 (S423+S425) ab52903 (Abcam), collagen type I OAMA03716, collagen type III OASB02204 (Aviva), vimentin #M7020 (Dako Cytomation) and HRP-coupled secondary antibodies (GE Life Sciences, Thermo Fisher Scientific). Membranes were treated with Western Lightning Enhanced Chemiluminescence Substrate (Perkin Elmer), and the chemiluminescence signal was recorded by the chemiluminescence reader Fusion FX6 (Vilber-Lourmat). Signals were quantified using densitometric analysis in ImageJ software.

Tissue sections (5 μm thick) were deparaffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was performed with proteinase K (20 μg/mL in PBS) for 30 minutes at 37°C. Samples were blocked with 5% bovine serum albumin/PBS and incubated overnight at 4°C with primary antibodies diluted in 5% bovine serum albumin/PBS [IL-1R1, ab106278 (Abcam, Cambridge UK); IL-6, AF-206 at 10 μg/mL (R&D Systems, Minneapolis, MN); vimentin, M7020 at 62 μg/mL (Dako)]. Slides were washed with PBS-Tween (0.05%) and incubated with anti-rabbit tetrarhodamine isothiocyanate (T6778; Sigma-Aldrich) and anti-mouse fluorescein isothiocyanate (F2012; Sigma-Aldrich) diluted in 5% bovine serum albumin/PBS for 1 hour at room temperature. Slides were washed with PBS-Tween and mounted using mounting medium with DAPI (H1200; Vector Laboratories). Images were acquired on a Nikon A1R point scanning confocal microscope.