Live Cell Imaging of Muller Glial Nuclear Migration During Zebrafish Retinal Regeneration

Begin with an agarose-embedded dorsal retinal explant from a transgenic zebrafish, mounted in a fluorodish with media.

The retina exhibits light-induced photoreceptor damage and contains Muller glial cells or MGCs with nuclei expressing GFP.

Place the dish into a multiphoton microscope chamber.

Using bright-field illumination, focus on the retina.

Switch to fluorescence mode to locate the MGCs.

Configure a z-stack from the ganglion cell layer or GCL to the outer nuclear layer or ONL.

Start live imaging.

Damaged photoreceptors release TNF-α, activating the MGCs.

MGC nuclei migrate basally within the inner nuclear layer or INL to replicate their DNA.

Then, the nuclei migrate apically to the ONL to undergo mitosis, producing neuronal progenitors.

After mitosis, the MGC nuclei and progenitors migrate basally back to the INL.

The progenitors proliferate, migrate to the injury site, and differentiate into photoreceptors, enabling retinal regeneration.

Analyze the captured images to visualize nuclear migration.

In the image acquisition software, open A1 MP GUI, TiPad, A1 Compact GUI, and ND Acquisition windows. For multiphoton imaging, ensure that the IR NDD Option is chosen in A1 Compact GUI window.