To begin, set up parental zebrafish with the desired single transgenes in standard mating cages the evening before mating. Using a divider in the mating cage, keep males and females separated until mating. Remove the divider in the morning of the next day to initiate mating.
At noon, at the latest, remove parental zebrafish from the breeding tanks. Strain the water from the mating cage through a sieve to collect the embryos. Then, place the embryos in a 100-millimeter diameter dish, containing E3 media, and keep them in an incubator at 28-degrees Celsius.
Afterward, remove unfertilized eggs and dead embryos from the dish using a plastic pipette and a stereo microscope. Using a three-milliliter plastic Pasteur pipette, transfer the four-day post-fertilization larvae with reporter transgene expression to a glass-bottom microwell dish. With the same pipette, remove the excess E3 media.
Now, take the melted low-melting agarose from the water bath or thermo block. Add a cooling drop of low-melting agarose to the dish, covering the glass bottom where the larvae were positioned. Place the larvae into the desired lateral position before the agarose solidifies.
Once the agarose solidifies, add E3 media containing a maximum of 0.02%MS-222 to the dish to avoid drying of the agarose.
