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The Use of Complementary Luminescent and Fluorescent Techniques for Imaging Ca2+ Signaling Events During the Early Development of Zebrafish (Danio rerio)

  • Sarah E. WebbEmail author
  • Andrew L. Miller
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)

Abstract

We have visualized many of the Ca2+ signaling events that occur during the early stages of zebrafish development using complementary luminescent and fluorescent imaging techniques. We initially microinject embryos with the luminescent Ca2+ reporter, f-holo-aequorin, and using a custom-designed luminescent imaging system, we can obtain pan-embryonic visual information continually for up to the first ~24 h postfertilization (hpf). Once we know approximately when and where to look for these Ca2+ signaling events within a complex developing embryo, we then repeat the experiment using a fluorescent Ca2+ reporter such as calcium green-1 dextran and use confocal laser scanning microscopy to provide time-lapse series of higher-resolution images. These protocols allow us to identify the specific cell types and even the particular subcellular domain (e.g., nucleus or cytoplasm) generating the Ca2+ signal. Here, we outline the techniques we use to precisely microinject f-holo-aequorin or calcium green-1 dextran into embryos without affecting their viability or development. We also describe how to inject specific regions of early embryos in order to load localized embryonic domains with a particular Ca2+ reporter. These same techniques can also be used to introduce other membrane-impermeable reagents into embryos, including Ca2+ channel antagonists, Ca2+ chelators, fluorescent dyes, RNA, and DNA.

Key words

Luminescent and fluorescent Ca2+ reporters Microinjection Holo-aequorin Calcium green-1 dextran Zebrafish embryos 

Notes

Acknowledgments

This work was supported by the Hong Kong Research Grants Council (RGC) General Research Fund awards 16101714 and 16100115 and the ANR/RGC joint research scheme award A-HKUST601/13. We also acknowledge funding support from the Hong Kong Innovation and Technology Commission (ITCPD/17-9). We thank Andrew Ho for helping us to photograph the equipment shown in Figs. 2 and 3.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Division of Life Science and State Key Laboratory of Molecular NeuroscienceHKUSTClear Water BayPeople’s Republic of China

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