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

Place a culture dish with dechorionated zebrafish embryos embedded in agarose over a microscope stage.

Take a dye solution containing a low concentration of antibody conjugates, carrying a red fluorescent-labeled antibody and an anti-DeltaD antibody.

Inject the dye solution into the embryo's hindbrain ventricle, targeting actively dividing radial glia progenitors or RGPs.

Release the embedded embryo and recover it in a nutrient-rich medium.

During recovery, fluorescent conjugates interact with a few Notch ligand DeltaD molecules on RGP, initiating internalization and endosome formation.

The low conjugate concentration allows the unbound DeltaD ligands to interact with the Notch receptor, activating the Notch signaling pathway in RGPs.

After recovery, transfer an anesthetized embryo with agarose to a glass dish with its dorsal side downwards for imaging. 

Perform time-lapse fluorescence imaging to capture the cell division, which shows an asymmetric partition of DeltaD-containing fluorescent endosomes into two daughter cells, confirming the activation of Notch/DeltaD signaling.