Embryos injected with photoactivatable MOs and/or lineage tracers were globally or regiospecifically irradiated as described.27 (link) For global irraditions, embryos were placed in a single-well of a 6-well microplate containing E3 medium, agitated continuously by vortexing, and irradiated for 15 minutes using a 365–370 nm light-emitting diode (LED) (Stanford Photonics) at an intensity of 10.6 mW/cm2. For regiospecific spot irradiations, chorionated 6-hpf embryos were arrayed in an agarose template (560-μm × 960-μm wells) filled with E3 medium, oriented with the ventral margin facing upward. The embryos were then irradiated using a Leica DM4500B upright compound microscope equipped with a mercury lamp, an A4 filter (Ex: 360/40 nm), and a 20x/0.5 NA water-immersion objective. An iris diaphragm was used to limit the targeted region to a 100-μm-diameter circle positioned approximately five cell layers above the margin, and the light intensity at the focal point was measured to be 40.9 mW/cm2. Embryos injected with cFD alone or in combination with the tbx16 cMO were irradiated for 15 seconds in this manner, and embryos injected with Kaede-NLS mRNA alone or in combination with the tbx16 cMO were irradiated for 60 seconds. Phenotype statistics for experiments using these photoactivatable probes are provided in Supplementary Table 3 .
Dm4500b upright compound microscope
Manufactured by Leica
The DM4500B is an upright compound microscope manufactured by Leica. It is designed for various laboratory applications. The microscope features optical components that enable high-resolution imaging and observation of samples.
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2 protocols using dm4500b upright compound microscope
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Photoactivation of Embryonic Lineages
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Photoactivation of Embryonic Lineages
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Embryos injected with photoactivatable MOs and/or lineage tracers were globally or regiospecifically irradiated as described.27 (link) For global irraditions, embryos were placed in a single-well of a 6-well microplate containing E3 medium, agitated continuously by vortexing, and irradiated for 15 minutes using a 365–370 nm light-emitting diode (LED) (Stanford Photonics) at an intensity of 10.6 mW/cm2. For regiospecific spot irradiations, chorionated 6-hpf embryos were arrayed in an agarose template (560-μm × 960-μm wells) filled with E3 medium, oriented with the ventral margin facing upward. The embryos were then irradiated using a Leica DM4500B upright compound microscope equipped with a mercury lamp, an A4 filter (Ex: 360/40 nm), and a 20x/0.5 NA water-immersion objective. An iris diaphragm was used to limit the targeted region to a 100-μm-diameter circle positioned approximately five cell layers above the margin, and the light intensity at the focal point was measured to be 40.9 mW/cm2. Embryos injected with cFD alone or in combination with the tbx16 cMO were irradiated for 15 seconds in this manner, and embryos injected with Kaede-NLS mRNA alone or in combination with the tbx16 cMO were irradiated for 60 seconds. Phenotype statistics for experiments using these photoactivatable probes are provided in Supplementary Table 3 .
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