Las advanced fluorescence software

MSCs were fixed on microfluidics channels in 4% paraformaldehyde for 15 min, washed with PBS, and stored overnight at 4 °C. Slides were permitted to come to room temperature the next morning, sticky channel overlays removed by single edge razor blade, and cells permeabilized in PBS with 0.2% Triton X-100 for 5 min at room temperature. Cells were incubated with Image-iT FX signal enhancer (Thermo Fisher, I36933) for 30 min at room temperature. Rabbit anti-pFAK polyclonal antibody (1:100 dilution, anti-pFAK Y397, Cell Signaling 3283S) was applied for labeling activated FAK for 1 h at room temperature in 2.5% BSA-0.1% Triton X-PBS. Slides were then washed with PBS. Cells were incubated with a 1:1000 dilution of goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (Invitrogen, Cat. No. A11008) for 1 h and washed in PBS. Cells were subsequently incubated with 25 µM DRAQ5 for 30 min at room temperature. Coverslips were mounted with Prolong Gold (Invitrogen). Images were captured by a Leica TCS SP5 confocal microscope with a Leica 63 × oil objective lens (NA 1.4) and analyzed with LAS Advanced Fluorescence software (Leica) and ImageJ 1.50i (NIH) to measure fluorescence intensity of the green channel.