Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Begin with early developmental stage zebrafish embryos in embryo media.

Remove the chorion, the protective membrane surrounding the embryos, and transfer the embryos to a fresh culture plate containing media.

Remove the tail portion of the embryo, then separate the head and gently detach the eyes from the head.

The eyes contain the retina, a thin layer of tissue which houses the retinal ganglion cells. 

Transfer the eyes to a tube containing zebrafish cell culture media.

Mechanically dissociate the eyes to release the retinal ganglion cells and form a single-cell suspension.

Take coverslips placed in a culture plate containing zebrafish culture media.

The coverslips are coated with a synthetic polymer and an adhesion protein.

Transfer the cells onto the center of the coverslip and incubate.

Retinal ganglion cells attach to the coated surfaces, and further, these cells develop long projections called axons.

Retrieve zebrafish embryos from the incubator, and immerse embryos in a 35-millimeter tissue culture dish containing 70% ethanol, for 5 to 10 seconds to sterilize it. Using a transfer pipette, transfer embryos to the E2 Media #1 dish containing sterile E2 media to wash off excess