Encyclopedia of Experiments: Biology
A subscription to JoVE is required to view this content.
Transcript
- Transfer a tricaine anaesthetized fish onto a wet sponge with the dorsal side facing up. Place the sponge carrying the fish under a microscope. Position a micro-injection needle at a 45 degree angle from the body axis near the dorsal muscle.
Next, locate the spot in front of the dorsal fin that is not covered with scales where you can insert the needle without applying great force. Then gently insert the needle and slowly inject the compound of interest along with a marker such as phenol red into the muscle.
If the compound of interest contains genetic material, electroporate the cells by placing electrodes on either side of the injection site and administering the current. The current makes the muscle cells permeable and able to efficiently take up the genetic material. After the procedure, immediately transfer the fish into a recovery tank. In the following protocol, we will perform an intramuscular injection on an adult zebrafish to deliver a DNA vaccine against mycobacteriosis.
To keep the fish in a fixed position during injection, use a piece of sponge with a groove cut in the middle as padding. Soak the sponge thoroughly in system water and set it in a Petri dish. Using a plastic spoon, transfer an anesthetized zebrafish to the wet sponge with the fish's ventral side down in the groove. Under the microscope, place the needle at a 45 degree angle close to the zebrafish's dorsal muscle. Then find the small spot without scales in front of the dorsal fin and push the needle into the muscle.
If any resistance is felt, try an adjacent spot. Use a foot switch to gradually inject the plasmid solution into the muscle. Under the microscope, the phenol red should be visible as it enters the muscle tissue.
Electroporate the fish immediately after injection by placing the tweezer type electrodes at each side of the injection site. Press the Start button on the electroporator and give it six 40 volt, 50 millisecond pulses. Then gently transfer the fish to a recovery tank. Finally, after anesthetizing the fish according to the text protocol, use a UV light to see EGFP expression near the injection site.
