An Antibody Uptake Assay for Imaging Notch/DeltaD Signaling in Zebrafish Radial Glia Progenitors

In This Article

Abstract

Source: Zhao, X. et al., Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors. J. Vis. Exp. (2023)

This video demonstrates an antibody uptake assay to image Notch/DeltaD signaling in zebrafish radial glia progenitors. It involves mounting agarose-embedded dechorionated zebrafish embryos, injecting a dye solution targeting DeltaD ligands on RGPs, and imaging under a fluorescence microscope for asymmetric cell division confirming Notch/DeltaD signaling activation.

Protocol

1. Preparation of zebrafish embryos

Set up fish crossing tanks in the afternoon before 5:00 p.m., with one female wild-type fish and one male Tg [ef1a:Myr-Tdtomato] fish by using dividers to separate them in each tank.
Remove the dividers before 11:00 a.m. from all crossing tanks the next morning. Keep quiet and do not disturb the fish while they are mating. Spawning typically occurs within 30 min after removing the dividers. The fertilized eggs remain on the bottom of the tank.
1. Collect fertilized eggs from the tanks with a mesh filter. Transfer the eggs by washing them off into a Petri dish full of egg water....

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Results

Fish breeding and imaging workflow; confocal microscopy diagram with larval injection and imaging.
Figure 1: Flowchart of experimental steps. Transgenic zebrafish expressing the MyR-Tdtomato reporter are outcrossed with AB wild-type on day 1. On day 2, red fluorescent embryos are selected for microinjection, followed by time-lapsing imaging. The whole .......