2.8. Focal adhesion immunolabelling
To analyse focal adhesion, 2 x 103 cells were plated were plated on a coverslip as indicated in the datasheet for the “actin cytoskeleton and focal adhesion staining kit” (Merck (FAK100), Spain). Cells were permeabilized with 0.5% Triton-X 100 in PBS for 30 minutes and then incubated in blocking solution for 1 hour at RT, containing 1% BSA. The primary anti-vinculin antibody (1:500 dilution: Merck (90227), Spain) was applied overnight (4 ºC) and after washing (5 minutes) in three times, the cells were incubated for 1h at RT with TRITC-conjugated Phalloidin (1:1000, Merck (90228), Spain) and an AlexaFluor488 Donkey anti-Mouse secondary antibody (1:1,000 dilution; ThermoFisher (A21202), USA). Images were acquired with an MMI CellCut plus (Olympus, Japan).
To quantify focal adhesions immunolabelled with anti-vinculin, images were analysed with ImageJ software (USA) and the total number of vinculin adhesion points were quantified per cell. The cell cultures were performed in triplicate and a total of 10 cells per sample were analysed.