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

Published: April 30, 2024

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 bott…

Representative Results

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 experiment on the 2nd day takes about 8 h.

Disclosures

The authors have nothing to disclose.

Materials

35mm glass bottom culture dish  MatTek corporation P35GC-1.5-10-C
Air pressure injector  Narishige IM300
Anti-Mouse-IgG-Atto647N  Sigma-Aldrich 50185
CaCl2.2H2O  Sigma-Aldrich C3306
Capillaries, 1.2 mm OD, 0.9 mm ID, with filament World Precision Instruments 1B120F-6
CSU-W1 Spinning Disk/High Speed Widefield Nikin N/A Nikon Ti inverted fluorescence microscope with CSU-W1 large field of view confocal. 
Dumont Medical Tweezers Style 5 Thomas Scientific 72877-D
Flaming-Brown P897 puller Sutter Instruments N/A https://www.sutter.com/manuals/P-97-INT_OpMan.pdf
KCl Millipore 529552
MgSO4.7H2O Sigma-Aldrich M2773
Micromanipulators World Precision Instruments WPI M3301R
Mouse anti-Dld  Abcam AB_1268496
Mouse IgG blocking buffer from Zenon Thermofisher Scientific Z25008
NaCl Sigma-Aldrich S3014
Phenol red Sigma-Aldrich P0290
Stemi 2000   Zeiss  N/A
Tricaine Sigma-Aldrich E10521
UltraPureTM low melting point agarose  Invitrogen 16520050

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Cite This Article
An Antibody Uptake Assay for Imaging Notch/DeltaD Signaling in Zebrafish Radial Glia Progenitors. J. Vis. Exp. (Pending Publication), e22116, doi: (2024).

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