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.