Ab683

Cells were fixed by incubating with 4% paraformaldehyde/PBS for 1 h at room temperature, washed with washing buffer (0.1% Tween–20/PBS) for 1 h once, and washed/permeabilized with 0.1% Triton X-100/PBS for 1 h at room temperature twice. Cells were blocked with a blocking buffer (3% BSA/2% goat serum/PBS) for 1 h at room temperature. Cells were incubated with primary antibodies/blocking buffer (mouse smooth muscle myosin heavy chain (SM-MHC), Abcam ab683, 1:50; rabbit collagen I, Abcam ab3,4710, 1:100; rabbit collagen IV, Abcam ab6,586, 1:100) overnight at 4°C on shaker. Cells were washed with washing buffer for 1 h at room temperature three times, then incubated with secondary antibodies/Hoeschst dye/blocking buffer (Alexa 488 goat-antimouse, Invitrogen A1,1001, 1:300; rhodamine goat–antirabbit, Millipore AP132, 1:300; Hoeschst dye, Cell Signaling Technology 4,082S, 2 μg/mL) for 1 h at room temperature on shaker. For cytoskeletal staining, cells were incubated with Rhodamine-phalloidin (Sigma P1951, 1:100 in blocking buffer) for 1 h at room temperature. Cells were again washed with washing buffer for 1 h three times, followed by imaging using confocal microscope (20× oil immersion, Leica SP8 confocal system). Images were colored and overlaid using Fiji software.