HH 36 embryonic limb explants were cultured in vitro for 24 h with blebbistatin (25 μM) or vehicle control (0.5% DMSO). (a) LIVE/DEAD staining of live (green) and dead (red) cells (left panel; bar, 25 μm), SHG detection of collagen fibers (red) (middle panel; bar, 10 μm), and phalloidin-stained actin (green) and DAPI-stained nuclei (blue) (right panel; bar, 5 μm), for vehicle control- and blebbistatin-treated tendons. Cell viability and collagen fiber organization did not appear to be altered by blebbistatin treatment. Blebbistatin disrupted the actin cytoskeleton. (b) Blebbistatin treatment decreased tendon elastic modulus, measured using FV-AFM (p = 0.02).