Primary mouse anti vinculin antibody

Cells were grown in 8-well chamber slides (Thermo Scientific, Waltham,
MA) for 24 hours before fixation and antibody staining, as previously
described^{26 (link)}. Briefly,
cells were incubated with a 1:400 dilution of primary mouse anti-vinculin
antibody (Sigma, St. Louis, MO) for 1 hour before addition of anti-mouse
AlexFlour-488 conjugated secondary antibody. Cells were fixed with rhodamine
phalloidin, according to the manufacturer’s protocol (Thermo Scientific,
Waltham, MA) before mounting using DAPI mounting medium (Vector Laboratories,
Burlingame, CA). Images were taken using a Zeiss LSM 710 confocal
microscope.

Samples were fixed 1 hour and 12 hours post seeding with 4% paraformaldehyde (Santa Cruz Biotech) for 15 minutes. Cell were permeablized using 0.2% Triton X (Sigma) in dPBS. Blocking and fixation were done with a solution of 2% bovine serum albumin (Sigma), 0.3 M glycine (Sigma), 0.2% Tween-20 (Sigma) in dPBS for at least 30 minutes. Samples were incubated with a primary mouse-anti-vinculin antibody (1:200) (Sigma) overnight at 4°C then washed. Anti-mouse 633 secondary (1:100) (molecular probes) and Actistain-555 (1:500) (Cytoskeleton, Inc.) were incubated for 1.5 hours at room temperature then washed. The nucleus was stained with DAPI (Molecular Probes) for 5 minutes and washed. Samples were inverted onto coverslips just before imaging. Images were acquired using the Zeiss 710 Axio-Observer with 40× oil immersion objective. For immunofluorescence imaging of fibroblast cells on TCPS ablation patterns, a 40× water immersion objective was used.