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Antibody Uptake Assay in the Embryonic Zebrafish Forebrain to Study Notch Signaling Dynamics in Neural Progenitor Cells In Vivo

Protocol
Part of the Methods in Molecular Biology book series

Abstract

Stem cells can generate cell fate heterogeneity through asymmetric cell division (ACD). ACD derives from the asymmetric segregation of fate-determining molecules and/or organelles in the dividing cell. Radial glia in the embryonic zebrafish forebrain are an excellent model for studying the molecular mechanisms regulating ACD of stem cells in vertebrates, especially for live imaging concerning in vivo molecular and cellular dynamics. Due to the current difficulty in expressing fluorescent reporter-tagged proteins at physiological levels in zebrafish for live imaging, we have developed an antibody uptake assay to label proteins in live embryonic zebrafish forebrain with high specificity. DeltaD is a transmembrane ligand in Notch signaling pathway in the context of ACD of radial glia in zebrafish. By using this assay, we have successfully observed the in vivo dynamics of DeltaD for studying ACD of radial glia in the embryonic zebrafish forebrain.

Keywords

Antibody uptake assay Asymmetric cell division Live imaging Notch signaling Radial glia Stem cell Zebrafish 

Notes

Acknowledgments

This work was supported by NIH (R01 NS095734) and Fudan Bio-elite program. We thank Xiang Zhao for discussions on the antibody uptake assay and comments on the manuscript, as well as Maximilian Fürthauer for sharing experience on the antibody uptake assay in the zebrafish spinal cord.

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.State Key Laboratory of Genetic Engineering, Department of Genetics, School of Life SciencesFudan UniversityShanghaiChina
  2. 2.Department of Bioengineering and Therapeutic Sciences, Programs in Human Genetics and Biological Sciences, ELi and Edythe Broad Center of Regeneration Medicine and Stem Cell ResearchSan FranciscoUSA

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