Dextran dmnb caged fluorescein biotin

A unit of 2 μg μl⁻¹ of dextran DMNB-caged fluorescein biotin 10,000 molecular weight (synthesized by Invitrogen using funding from NIH R01 grant #DK078209-04S1, Tomoko Obara) was injected into embryos at the one-cell stage. Injected embryos were grown to ∼85% epiboly, dechorionated in 0.5 mg ml⁻¹ pronase from Streptomyces griseus (Roche Diagnostics), washed three times in E3 embryo medium, then individually placed into wells on a Petri dish containing a bed of 1% agarose. Embryos were orientated to a lateral position and fitted to the 306-point grid. Uncaging was performed on the left side of all embryos by selecting a co-ordinate for a 4-s exposure to a 405-nm diode laser using a Sim Scanner on an Olympus FluoView FV1000 confocal laser scanning microscope. As the laser passes through all layers of the embryo, and out the contralateral side, both the epiblast and hypoblast are labelled on either side of the embryo. Uncaged embryos were then imaged and placed in fresh E3 embryo medium for further experimentation. Only the uncaged cells on the left-hand side of the embryo were used for the fate mapping analysis.

2 μg/μl of dextran DMNB-caged fluorescein biotin 10,000 MW (Invitrogen Tomoko Obara NIH grant# R01 DK078209-04S1) was injected into embryos at the one-cell stage. Injected embryos were grown to the 12-somite stage, manually de-chorionated and placed into a well on a petri dish containing a bed of 1% agarose. Embryos were oriented dorsal side up and un-caging was performed by selecting a region encompassing somite 8 and the intermediate mesoderm lateral to this somite for a 10 second exposure to a 405 nm diode laser using a Sim Scanner on an Olympus FluoView FV1000 confocal laser scanning microscope. Un-caged embryos were placed in fresh E3 embryo medium and grown to 24 hpf. To detect un-caged fluorescein, the in situ hybridization protocol was followed and anti-fluorescein fragments (Roche Diagnostics) were used to label the un-caged fluorescein.