Dna stain dapi

For immunocytochemistry of active integrin‐β1, cells were seeded on sterile coverslips in a six‐well plate at a density of 173,000 cells per well. The coverslips were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X‐100 on ice for 1 min. After blocking in 5% BSA‐1X PBS, active integrin‐β1 antibody (1:100) was added for 1 h followed by 1h incubation with secondary antibody. After repeated washing in 1X PBS, cells were stained with the DNA stain DAPI (4,6‐diamidino‐2‐phenylindole dihydrochloride; #1023627001; Roche) and mounted using Prolong gold anti‐fade mount media (#P36930; Invitrogen) on glass slides. Imaging and z‐stacks (1‐µm z‐slice) were acquired using a Leica TCS SPEII confocal microscope maintaining consistent acquisition parameters between experiments.

For immunocytochemistry of FN, SMA, or COL, IMR90 cells were seeded on sterile coverslips in a 6-well plate at a density of 80,000 cells per well. The coverslips were fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 for 1 min on ice. The cells were then blocked with 5% BSA-1x PBS for 1 h on ice. After blocking, FN antibody (1:70), SMA (1:1000) or COL (1:100) was added for 1h followed by 1h incubation with a secondary antibody. After secondary incubation, the cells were washed repeatedly with 0.2% Tween in 1xPBS. After washes, the cells were stained with the DNA stain DAPI (4, 6 diamidino-2-phenylindole dihydrochloride) (Roche, #1023627001) and mounded with Prolong gold anti-fade mound media (Invitrogen, #S36936) on glass slides. Images and z-stacks (1μM z-slice) were acquired using a Leica TCS SPEII confocal microscope at consistent acquisition parameters for each experiment.