Glass coverslips were coated with 10 μg/ml collagen I (BD Biosciences) at 4 °C overnight, then rinsed three times with PBS. Cells were plated on coverslips in normal culture medium and allowed to adhere for 4 hours before fixation. Cells were fixed, permeabilized, and immunostained as described previously57. Briefly, cells were fixed for 15 minutes with 4% paraformaldehyde containing 10 mM PIPES, pH 6.8, 2 mM EGTA, 2 mM MgCl2, 7% sucrose and 100 mM KCl for 15 min at room temperature, and permeabilized with 0.25% Triton X-100. Cells were blocked for 1 hour with 3% BSA + 1% goat serum in PBS. The following primary antibodies were used at indicated concentrations and incubated at 4 °C overnight: rat anti-CD104 (439-9B, BD Pharmingen, 1:100) mouse anti-Laminin-5 (γ2 chain, clone D4B5, Millipore, 1:500 dilution) in 3% BSA in PBST overnight at 4 °C. Cy3-conjugated goat anti-rat and Cy2-conjugated goat anti-mouse (Jackson Immuno Research, 1:500) and Alexa Fluor 647 phalloidin were incubated with 3% + 1% goat serum BSA for 1 hour at room temperature in dark. Coverslips were mounted on glass slides using 50% glycerol solution and sealed with clear polish. Images were acquired by total internal reflection (TIRF) microscopy using a Nikon Eclipse Ti. Images were processed for colocalization analysis and Pearson’s correlation coefficient by NIS Elements AR 3.2 software.