Tritc or alexa fluor 488 conjugated phalloidin

Cells were seeded onto human fibronectin (10 µg/ml; Sigma-Aldrich)-coated coverslips. After transfection or incubation overnight, cells were fixed in 4% paraformaldehyde in PBS for 20 min at RT and subsequently permeabilized with 0.2% Triton X-100 in PBS for 5 min. For F-actin staining, cells were incubated with either TRITC- or Alexa Fluor 488–conjugated phalloidin (Invitrogen) and diluted in PBS for 1 h at RT. After this incubation, cells were washed three times in PBS. For detection of paxillin, primary antibodies were diluted in PBS with 3% BSA (Thermo Fisher Scientific) and incubated for 2 h at RT. After labeling with the primary antibodies, cells were washed three times with PBS before incubation with either Alexa Fluor 568– or 488–conjugated secondary antibodies (Invitrogen) and phalloidin. Cells were then imaged using either a microscope (IX71; Olympus) with a 40× oil-immersion objective (UPlanFLN) with a numerical aperture of 1.3 and Image-Pro Plus software (Media Cybernetics) or a confocal laser-scanning microscope (LSM510; Carl Zeiss) with a Plan Fluorite 100× oil objective with a numerical aperture of 1.45 and the accompanying LSM510 software. Focal adhesion number and length were quantified using ImageJ software (National Institutes of Health).