Imaging Embryo Development with Confocal Microscopy

Embryos at the ten-somite stage were mounted in 0.8% low-melting agarose and imaged at 25 °C using a laser scanning confocal microscope (LSM 710, Carl Zeiss) running the software Zen 2012 sp5 (Carl Zeiss). Confocal images of the region of interest in ubiquitous or mosaic membrane-labelled embryos were taken either at 2.5 s intervals using a ×40 water immersion objective (LD C-Apochromat 1.1 W, Carl Zeiss) or through a 3D timelapse with 1.0 µm optical sections every 16 s using a ×25 water immersion objective (LD C-Apochromat 1.1 W, Carl Zeiss). Imaging of ferrofluid droplets in the embryo was done as previously described^{28 (link)}. To visualize myosin and actin dynamics at the cell–cell contact over time, confocal images of Tg(actb2:MA-mCherry2)hm29 × Tg(actb2:myl12.1-EGFP) and Tg(actb2:MA-mCherry2)hm29 × Tg(actb1:GFP-Has UTRN)e116) double transgenic embryos, respectively, were taken in the region of interest at 2 s intervals using a ×40 water immersion objective. Ferrofluid droplets were labelled using a custom-synthesized fluorinated rhodamine dye^{58 (link)}, which was diluted in the ferrofluid oil at a final concentration of 37 μM.