Immunofluorescence Staining of Focal Adhesions

Similar number of cells were seeded on 10 μg/mL fibronectin-coated (Roche, USA) or 10 μg/mL laminin-coated (Sigma-Aldrich, USA) coverslips for 6 h to allow for single cell imaging. Samples were fixed with 4% formaldehyde (Sigma-Aldrich, USA) for 20 min at room temperature, permeabilized in 0.2% Triton-X-100 (Sigma-Aldrich, USA) for 10 min at room temperature, and blocked with 4% bovine serum albumin (Sigma-Aldrich, USA) for minimum 30 min. Cells were then incubated with primary antibodies in blocking buffer at 4 °C overnight, followed by secondary antibody incubation for 1 h at room temperature. Stress fibers were stained with phalloidin conjugated with Alexa Fluor 546/488 (Invitrogen, USA). Coverslips were mounted using VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, USA). Images were acquired using Zeiss LSM 710/980 confocal microscopes (Carl Zeiss, Germany) and analyzed with FIJI software (NIH, version 1.53)^{69 (link)}. Co-localization analysis was carried out with Coloc2 plugin in FIJI software (NIH, version 1.53c)^{69 (link)} with the segmented FA as the mask^{34 (link)}.