The Use of Complementary Luminescent and Fluorescent Techniques for Imaging Ca2+ Signaling Events During the Early Development of Zebrafish (Danio rerio).
Methods Mol Biol
; 1929: 73-93, 2019.
Article
in En
| MEDLINE
| ID: mdl-30710268
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
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Zebrafish
/
Aequorin
/
Calcium Signaling
/
Lissamine Green Dyes
Limits:
Animals
Language:
En
Journal:
Methods Mol Biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2019
Document type:
Article
Publication country:
EEUU
/
ESTADOS UNIDOS
/
ESTADOS UNIDOS DA AMERICA
/
EUA
/
UNITED STATES
/
UNITED STATES OF AMERICA
/
US
/
USA