C apochromat 40x 1.2 na objective

Dechorionated embryos were mounted dorso-laterally (as shown in Figure 1C) using a canyon mount cast in 1% agarose filled with 1X Danieau buffer as previously described in detail (Swinburne et al., 2018 (link)). Confocal z-stacks were acquired with an upright Zeiss LSM 710 laser scanning confocal microscope using a C-Apochromat 40X 1.2 NA objective for all fluorescence microscopy data, except Figures S3A, 6A, 6B, 6D and 6H, where Zeiss LSM 980 laser scanning confocal microscope with C-Apochromat 40X 1.2 NA objective and Airy Scan 2 module was used. For single time points, embryos were immobilized by soaking in 1% tricaine. For long-term time lapse imaging, embryos were immobilized with 500 μM α-bungarotoxin protein (aBt from Tocris) (Swinburne et al., 2015 (link)) injected into the heart ~30 minutes prior to imaging (at variable stages depending on the experiment). Time-lapse imaging took place in a home-built incubator at 28.5°C. In a typical experiment using Zeiss LSM 710, ~250 μm × 250 μm × 150 μm volume with a voxel size ~0.2 μm × 0.2 μm × 0.5 μm captured the entire OV within ~3 minutes. With the better resolution of the Zeiss LSM 980 Airy Scan 2, the voxel size improved to ~0.12 μm × 0.12 μm × 0.35 μm. The volume and speed of acquisition was adjusted depending on the experiment.