This article details a method for isolating retinal ganglion cells (RGCs) from zebrafish embryos. The protocol includes steps for embryo preparation, eye dissection, and cell culture to facilitate RGC growth and axon development.
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 ethanol. Then, transfer the embryos to the E2 Media #2 dish, and remove their chorions with sharp forceps. Finally, transfer embryos to the E2 Media #3 dish to perform dissections.
Using a pair of fine forceps, position and hold embryos anterior to the yolk with one of the forceps, and remove the tail posterior to the yolk sac with the other forceps. Grab the neck with forceps and take off the head to expose the brain and eyes to the E2 media. With the tip of fine forceps, gently roll the eyes from the head while holding the cranial tissue down with the second forceps. Transfer four eyes to one of the previously prepared tubes containing ZFCM+.
Gently triturate up and down with the P20 pipette about 45 times to dissociate cells. Transfer the ZFCM+ with the dissociated cells to the center of the coverslips. Maintain cultures on the benchtop at 22 degrees Celsius, on a polystyrene foam rack to absorb vibrations. On the day of imaging, check cells under the microscope to validate growth of RGC axons.
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