Microscope cage incubator

Fluorescence images were taken on a Nikon A1 confocal microscope with the Elements software package using a 100× Plan Apo λ (NA 1.45) oil objective. Excitation of fluorescence probes was made with solid‐state lasers and their emission isolated by bandpass filters as follows; SYTO40: 405 nm excitation, 425–475 nm filter, MTG: 488 nm excitation, 500–550 nm filter, FM 1–43: 488 nm excitation, 521.5–554.5 nm filter, FM 4–64: 488 nm or 561 nm excitation and 575–625 nm filter. Short‐term imaging was done on glass slides coated with 0.01% poly‐l‐lysine (Sigma‐Aldrich, St. Louis, MO, USA). Imaging over time was performed with an Attofluor cell chamber (ThermoFisher/Invitrogen, Waltham, MA, USA) using a standard poly‐l‐lysine‐coated coverslip as a base. For long‐term experiments, the sample was kept at a constant growth temperature of 30°C by an Okolab microscope cage incubator. TEM imaging was performed by the Ohio State University Ohio Agricultural Research and Development Center's Molecular and Cellular Imaging Center in Wooster, Ohio.

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.