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Mounted embryos were imaged in an upright Zeiss LSM 710 confocal microscope.
Mounted embryos were imaged on a BioRad Radiance 2100 confocal mounted to a Zeiss inverted microscope using a 40× or 63× objective.
Mounted embryos were sectioned at 10 µm using a Leica cryostat and used for immunofluorescence after air-drying for at least 1 hour.
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The mounted embryo was bathed with Medium 199 (Sigma) with 5% fetal bovine serum and 10% chick serum (culture medium M199) as previously described [40] and the Petri dish placed on a heated stage insert (PECON Tempcontrol 37) inside a custom built microscope incubator box assuring that the embryo, objective and stage were kept at 37 38°C during time-lapse image recording.
Photographs of whole mount embryos were taken with a Stereoscopic Zoom Microscope SMZ1000 (Nikon Corporation, Tokyo, Japan).
Ciona whole mount embryos were also mounted with Vectashield mounting medium with DAPI (Vectastain; Vector Laboratories, Burlingame, CA).
Whole-mount embryos were imaged with a Nikon SMZ 1500 stereo microscope.
For vibratome sections, whole-mount embryos were stored in 4% PFA after in situ hybridization.
In situ hybridization on whole mount embryos were carried out using standard protocols.
Whole mount embryos were imaged on a Leica stereomicroscope fitted with a Zeiss Axiocam color camera.
Whole-mount embryos were partially cleared in 50% glycerol and imaged using a stereomicroscope.
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