2.12. Vinculin/F‐actin immunofluorescence

Cells were seeded on a poly‐l‐lysine‐coated cover slip (Corning). After 48 h under 5% CO₂ at 37 °C incubation, the cells were fixed with 4% paraformaldehyde for 30 min and permeabilize with 2% Triton‐X‐100 for 15 min at RT. Blocking with 5% BSA + 2% Triton‐X‐100 for 1 h at RT was followed by co‐incubation with Alexa Fluor 594 Phalloidin (Thermo Fisher) and monoclonal anti‐vinculin antibodies (Sigma‐Aldrich) for 1 h at RT. Then, cells were washed 3× with PBS and incubated with goat anti‐mouse IgG (H + L) cross‐adsorbed secondary antibody, Alexa Fluor 488 (Invitrogen, Eugene) for 1 h at RT. After 3× washes with PBS, the cover slip was mounted on a slide with VECTASHIELD antifade mounting medium with DAPI. Images were acquired on Nikon Eclipse Ti‐U using the 40× objective.