Mouse monoclonal anti α sma primary antibody

Immunostaining on 2D hydrogels and tissue culture plastic (TCPS) was performed as described previously.³³ Briefly, cells were fixed with 4% paraformaldehyde in PBS for 20 minutes and were permeabilized with 0.1% Triton-X-100 (Fisher Scientific) in PBS. The samples were blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich) overnight to prevent non-specific staining. VICs were stained with mouse monoclonal anti-α-SMA primary antibody (Abcam, 1:200 dilution, ab7817) for one hour at room temperature. Samples were washed with PBS (3x) and incubated with the secondary antibody solution, comprising goat anti-mouse Alexa Fluor 488 (Life Technologies, 1:300 dilution), HCS Cell Mask (Life Technologies, 1:5000 dilution), and 4’−6-diamidino-2-phenyindole (DAPI, Life Technologies, 1:500 dilution). Samples were inverted onto a glass-bottom 24 well plate (Cellvis) and imaged on an Operetta high-content confocal microscope using a 20x objective (Perkin Elmer). VIC activation was quantified using Harmony software (Perkin Elmer) to count VICs expressing α-SMA stress fibers based on α-SMA intensity values normalized to Cell Mask intensity values. At least three fields of view were quantified per gel, with at least three gel replicates performed for each condition. Data are presented as mean ± standard error measurement.