Primary mouse monoclonal antibodies

HVOX (1.0 × 10⁵) were seeded on chamber slides and grown for 72 hours under each experimental condition. Cells were then fixed in 4% paraformaldehyde for 10 minutes and then permeabilized and blocked with a PBS solution containing .1% Triton-X and 5% normal goat serum. The slides were incubated at 4°C overnight with primary mouse monoclonal antibodies against αSMA (1:200; Sigma-Aldrich, St. Louis, Missouri, USA) and then the corresponding Alexa-Fluor 555 goat anti-mouse IgG (1:500; Invitrogen, Carlsbad, California, USA) secondary antibody. For negative controls, primary antibodies were omitted. Digital images were captured with a Nikon Eclipse Ni-U fluorescence microscope (Nikon Inc, Tokyo, Japan), and ImageJ Software (National Institutes of Health; v1.41) was used to count αSMA-positive cells and nuclei. Analyses were performed on 10 randomly chosen, 3.0 mm² fields for each condition under 10× magnification.

After 24h seeding, cultures were fixed with 4% paraformaldehyde in PBS (SIGMA-ALDRICH, St. Louis, MO) at RT for 20 min. Samples were blocked and permeabilized in 10% bovine serum albumin and 0.1% Triton-X 100 for 10 and 5 minutes, respectively, and afterwards incubated for 1h with primary mouse monoclonal antibodies directed against vimentin (1:200, SIGMA-ALDRICH, St. Louis, MO). After rinsing, the following stains were used: rhodamine-phalloidin (1:500, Molecular Probes, Eugene, OR) was used to label actin filaments, Hoechst (1:1000, Molecular Probes, Eugene, OR) was used to label cell nuclei and anti-mouse Alexa Fluor 488 secondary antibody (1:500, Molecular Probes, Eugene, OR) was used for vimentin filament visualization. The cells were examined using a Leica DMIRE2 inverted microscope (Leica, Buffalo Grove, IL) and images were acquired using a Hamamatsu camera (Hamamatsu, Japan). Immunofluorescence was recorded with a 100x oil lens with a numerical aperture of 1.25. In order to quantify cell adherent area, a 40x air lens with a numerical aperture of 0.60 and phase contrast was used. For each sample, a minimum of 20 images was acquired from different locations and approximately 100 cells per substrate were analyzed. Cell area was calculated using ImageJ software by precise tracing of single cell outlines (Image J Software, NIH, Bethesda, MD) ³³ .