Live embryos were collected prior to nc14, dechorionated, mounted on a permeable membrane, immersed in Halocarbon 27 oil, and put under a glass coverslip as in Garcia et al., 2013 (link). Individual embryos were then imaged on a Nikon A1R point scanning confocal microscope using a 60X/1.4 N.A. oil immersion objective and laser settings of 40uW for 488 nm and 35uW for 561 nm. To track transcription, 21 slice Z-stacks, at 0.5 um steps, were taken throughout the length of nc14 at roughly 30 s intervals. To identify the Z-stack’s position in the embryo, the whole embryo was imaged after the end of nc14 at 20x using the same laser power settings. Later in the analysis, each transcriptional spot’s location is described as falling into one of 42 anterior-posterior (AP) bins, with the first bin at the anterior of the embryo. Unless otherwise indicated, embryos were imaged at ambient temperature, which was on average 26.5°C. To image at other temperatures, embryos were either heated or cooled using the Bioscience Tools (Highland, CA) heating-cooling stage and accompanying water-cooling unit.
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