Myofibroblast Stress Fiber Visualization

Capturing myofibroblast stress fiber formation using immunofluorescence was accomplished similar to previous studies [36 (link)] by growing 1.75 × 10⁵ cells/well on glass coverslips in 6-well plates and treated as described in the scratch assay method above. Before immunofluorescent staining each well was washed 2x with PBS (5 minutes/wash), fixed with 4% paraformaldehyde in PBS (5 minutes at room temperature), washed again with PBS (2× 5 minutes), permeablized with 0.1% Triton X-100 in PBS (3 minutes at room temperature), and washed a final time in PBS (2× 5 minutes). Cells were then stained for both filamentous actin (1:40 dilution; 1 hour at room temperature) with tetrarhodamine isothiocyanate (TRITC)–labeled phalloidin (SC-301530; Santa Cruz) and nuclei with 4′,6-diamidino-2-phenylindole (1:500 dilution, DAPI; Molecular Probes, Eugene, OR). Coverslips were mounted onto slides using SlowFade Gold Antifade Mountant (S36937; ThermoFisher Scientific, Waltham, MA) and images collected on a Zeiss LSM 780 confocal system (Carl Zeiss, Jena, Germany) with a 40x water immersion objective with the same florescent parameters for all images. Assays were completed in technical triplicate and images relative to the average shown.