Dmi8 confocal microscope

Cells were fixed with 4 % formaldehyde or ice cold methanol (ROCK1), followed by quenching of autofluorescence with 100 mM glycine, and blocking of unspecific antibody binding with 1 % BSA. Antigens were labeled with primary antibody in 0.1 % BSA (anti-E-Cadherin (1:800, Invitrogen), anti-myosin IIb (1:200, Cell Signaling), anti-ROCK1 (1:300, ab134181, abcam,) at 4 °C overnight, followed by detection with secondary, fluorochrome labeled antibodies (1:250 in 0.1 % BSA, Invitrogen). F-actin was labeled using phalloidin -TRITC (1:200, Invitrogen). Nuclei were labeled with DAPI (Invitrogen). Specimens were imaged using a Zeiss Observer Z1 inverse microscope, a Leica DMi8 confocal microscope, or a Zeiss 880 / airyscan. Image analysis was performed in ImageJ 1.49 / Fiji 1.0 as previously described [17 (link)]. Briefly, membranes and cytoplasms of each cell were manually gated and mean signal intensities determined. Ratios of the membrane and the cytoplasmic signal were calculated, and analyzed and plotted using GraphPad Prism 7. Scale bars apply to all images in each panel.