Embryo Microinjection: A Technique to Deliver a Compound into the Zebrafish Yolk

- Place two female and two male fish into a divided breeding tank one night before the injection. The following morning, remove the divider and let the fish breed until you observe eggs at the bottom of the tank. Collect the eggs and wash them with E3 medium containing methylene blue, a fungicide. Observe the eggs under a microscope and remove unfertilized eggs. Unfertilized eggs have a clear yolk membrane, while fertilized eggs have a dark yolk membrane.

Keep 10 embryos in a separate Petri dish as uninjected controls. Using a transfer pipette, align the embryos into the trough of a microinjection plate at room temperature and place it under the microscope. Gently push the microinjection needle through the chorion into the yolk and slowly inject a compound of interest containing a marker such as phenol red. Cytoplasmic flow allows the compound to diffuse into the embryonic cells.

After injection, grow the embryos in E3 medium with methylene blue at 28 degrees Celsius. Keep checking the health of the embryos. Remove any dead or abnormally developing embryos and change the medium daily. In the following protocol, we will use microinjection to deliver a ribonucleoprotein, or RNP compl