Fluorophores were ubiquitously expressed at 25C using [P{tubP-GAl4}LL7 or P{w[+mC]=Act5C-GAL4}25FO1] and the adult progeny were examined for eye morphogenesis defects and reduced viability (% of animals carrying the fluorophore transgene relative to a balancer).
Imaging in zebrafish. Adult zebrafish, both TL and AB wild-type strains, were maintained under standard laboratory conditions. Expression plasmids pCS2-mIFP-H2B and pCS2-HO1 were created by PCR amplification of mIFP-H2B and HO1 ORF's, respectively, and then cloned into pCS2+. Capped messenger RNA was synthesized using the mMESSAGE mMACHINE SP6 kit (Ambion). 100 pg of mIFP-H2B mRNA with 100 pg of HO1 mRNA were injected at the one-cell stage. Fluorescent and brightfield images were taken at 30 h.p.f. with a charge-coupled device camera (Andor; Clara DR-2199) on the Nikon Eclipse Ti microscope. Manually dechorionated embryos were embedded in 1.5% low-melt agarose within glass-bottom Petri dishes (MatTek Corporation). Eye-specific images were taken with a 40×/1.3 NA Plan-Fluor oil objective. mIFP fluorescence was imaged with a 638nm laser line and a redshifted Cy5.5 filter. Z stacks of 2.5 μm intervals were acquired for each magnification.
Imaging in zebrafish. Adult zebrafish, both TL and AB wild-type strains, were maintained under standard laboratory conditions. Expression plasmids pCS2-mIFP-H2B and pCS2-HO1 were created by PCR amplification of mIFP-H2B and HO1 ORF's, respectively, and then cloned into pCS2+. Capped messenger RNA was synthesized using the mMESSAGE mMACHINE SP6 kit (Ambion). 100 pg of mIFP-H2B mRNA with 100 pg of HO1 mRNA were injected at the one-cell stage. Fluorescent and brightfield images were taken at 30 h.p.f. with a charge-coupled device camera (Andor; Clara DR-2199) on the Nikon Eclipse Ti microscope. Manually dechorionated embryos were embedded in 1.5% low-melt agarose within glass-bottom Petri dishes (MatTek Corporation). Eye-specific images were taken with a 40×/1.3 NA Plan-Fluor oil objective. mIFP fluorescence was imaged with a 638nm laser line and a redshifted Cy5.5 filter. Z stacks of 2.5 μm intervals were acquired for each magnification.