Em charge coupled device camera

Fixed cells were imaged using an inverted research widefield microscope (Eclipse Ti; Nikon) with perfect focus system, equipped with a 60×1.49 NA Apochromat total internal reflection fluorescence (oil) objective lens, a microscope cage incubator (OkoLab), and an EM charge-coupled device (CCD) camera (Andor Technology) controlled with NIS-Elements Ar 4.0 software (Nikon). All widefield images, unless specifically indicated otherwise were background subtracted by ImageJ's rolling ball (r=20) and sharpened for display by Fiji/ImageJ's unsharp mask filter (r=1, weight=0.6).
Fixed embryos were imaged on the same microscope using the Nikon C1 Confocal system. For time-lapse imaging, embryos were dechorionated at 50% epiboly stage and embedded in 0.3% agarose in E3 embryo medium. DIC images were taken every 15 min on the same microscope using a 10×0.3 NA Plan Fluor objective lens (Nikon). Embryo staging was determined by wild-type embryos imaged simultaneously with mutants or morphants. Live embryos at 12-24 hpf and fixed embryos after whole-mount in situ hybridization were imaged using a Leica MZFLIII upright microscope.

For IF stainings, cells were plated on coverslips coated with either Fibronectin (HUVECs) or Collagen (MDCKs). Prior to fixation with 2% paraformaldehyde for 20 minutes, MDCK cells were treated with either HGF or blebbistatin (Fig. 6 and figure S1). After fixation, cells were permeabilized with 0,4% Triton X-100 for 5 minutes and blocked in 2% BSA for 1 hour. Phalloidin, primary- and secondary antibodies were diluted in 2% BSA and incubated with the cells for 1 hour. Afterwards, cells were mounted in Mowiol 4–88/DABCO solution (Sigma-Aldrich). For live imaging, lentivirally transduced HUVECs were plated into Lab-Tek chambered 1.0 boro-silicate coverglass slides coated with fibronectin and cultured in EBM-2 medium supplemented with EGM-2 bulletkit. Live (at 37 °C) and fixed cells were imaged using an inverted research widefield microscope (Eclipse Ti; Nikon) with perfect focus system, equipped with a 60 × 1.49 NA Apochromat total internal reflection fluorescence (oil) objective lens, a microscope cage incubator (OkoLab), and an EM charge-coupled device (CCD) camera (Andor Technology) controlled with NIS-Elements Ar 4.0 software.