Cell fixation and immunofluorescence

Glass coverslips were treated in 70% HNO₃ (nitric acid) for 30 min before washing with a copious amount of water. Coverslips were then coated with 1 mg/ml of fibronectin (#F1141; Sigma-Aldrich) for 2 h and washed with PBS. Cells were seeded on coverslips and left to settle for 4 h before being fixed in 4% PFA. Cells were permeabilized with buffer containing 20 mM glycine and 0.05% Triton X-100 in PBS for 5 min before being incubated with primary antibody (1:200 dilution) and then secondary antibodies (1:200 dilution), phalloidin (1:100 dilution), and DAPI (1:1,000 dilution). Cells were then mounted on a glass slide using ProLong Diamond Antifade Mountant (#P36961; Invitrogen).