Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

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.