Immunofluorescence analysis of focal adhesions

Twenty-four hours after transfection, cells were detached, diluted 1:3 and seeded on glass cover slips. The next day, growth medium was removed and cells washed twice in cold PBS and fixed with 4% paraformaldehyde (VWR International, Radnor, PA, USA) in PBS for 10 min at room temperature. Cover slips were incubated with a permeabilization solution (PBS/Triton X-100 0.2%, v/v), blocked for 1 h with a blocking solution (PBS/normal goat serum 5%, v/v, FBA 1%, v/v) and then incubated over night at 4 °C with anti-phospho-FAK Y397 (pFAK-Y397) (Cell Signaling #3283, 1:200; RRID:AB_10891442) and anti-Vinculin (Sigma-Aldrich, V9131, 1:500; RRID:AB_477629). Cells were then washed with cold PBS and incubated for 1 h with fluorophore-conjugated secondary antibodies (respectively AlexaFluor 568, RRID:AB_143157, and 647, RRID:AB_2535814). Nuclei were stained with 1 μg/ml Hoechst 33342 (Thermo Fisher Scientific, 62249, 1:1000) and F-actin was stained with fluorescein phalloidin (Thermo Fisher Scientific, F432, 1:100) for 30 min at room temperature. Confocal microscopy images were acquired by a LSM800 microscope (ZEISS; RRID:SCR_015963) equipped with an oil-immersion 63X objective and ZEN lite 2012 software Service Pack 2 (RRID:SCR_018163). Fluorescence images were deconvoluted using the software Huygens Professional engine 20.10.0p2 (Scientific Volume Imaging B.V.; RRID:SCR_014237) and adjusted for brightness, contrast and color balance by using Fiji (RRID:SCR_002285) analysis software (based on ImageJ v- 2.1.0/1.53c). The images represent projections and were achieved by summing the fluorescence signal of central z-stacks (two planes, 0.3 µm). The analysis of pFAK1-containing focal adhesions was performed by a modified version of the protocols previously published^25,58 using Fiji imaging software. Briefly, the two central z-stacks of confocal images were summed, then pFAK-Y397 and Vinculin signals were merged in order to determine pFAK-Y397 containing focal adhesions. Pictures were thresholded using MaxEntropy algorithm, and particles with a size >0.1 µm² were analyzed. Nuclear/perinuclear areas were excluded from the analysis. Area, perimeter, maximum Feret’s diameter of each pFAK-Y397-containing focal adhesion was analyzed and shape factor was calculated using the following formula: