Intramuscular (IM) Injection: Compound Delivery into the Dorsal Muscle of an Adult Zebrafish

1. Injection of the DNA Vaccine and Electroporation

1. For immunizations according to this protocol, use healthy, 6 to 12 month-old adult zebrafish. Keep the fish in a flow-through system with a 14/10 h light/dark cycle, with a maximum of seven fish per 1 L of water.
2. Prepare a 0.02% 3-aminobenzoic acid ethyl ester (tricaine) solution (pH 7) in tank water for anesthetizing the fish. Use a 10 cm Petri dish or something similar.
3. Prepare a recovery tank by filling a 5 L beaker with 3 L of clean system water.
4. Prepare a vaccination padding to keep the fish in a fixed position during the vaccination. Take a 5 x 7 cm² piece of a 2 to 3 cm-thick sponge. Cut a groove into the sponge with a scalpel blade or sharp scissors.
   NOTE: The same vaccination padding can be used in multiple experiments. Disinfect the sponge between the experiments by soaking it in 70% ethyl alcohol and allow to dry.
5. Thoroughly soak the sponge in the system water and set the sponge on a Petri dish.
6. Fast the fish 24 h before the vaccinations.
7. Anesthetize one zebrafish by placing it on a Petri dish containing 0.02% tricaine. Wait until the fish does not respond to touch stimulation and until there is no movement of the gills. Anesthetize a single fish at a time.
8. Using a plastic spoon, transfer the anesthetized zebrafish onto the wet sponge and set the fish’s ventral side down into the groove. In the correct position, ensure that the head and most of the body of the fish are inside the groove and the dorsal fin and the tail are protruding out from the groove.
9. Under the microscope, carefully place the needle in an approximately 45° angle close to the zebrafish’s dorsal muscle, using the x- and y-axis fine-tuning wheels on the micromanipulator.
10. Find the small spot without scales in front of the dorsal fin, where pushing the needle does not demand force. If resistance is felt, try an adjacent spot. Avoid injuring the spine, the dorsal fin, or the scales.
    NOTE: If the needle bends while pushing, shorten the needle slightly by cutting it.
11. Use the foot switch to gradually inject the vaccine solution into the muscle. Observe the injection through the microscope: phenol red is visible as it enters the muscle tissue. Adjust the duration of the pulse if needed.
    NOTE: Alternatively, use the pulse initiator button on the front panel of the pneumatic pump for the injection. However, the use of a remote foot switch allows using the other hand for adjusting the pulse length by the 10-turn dial wheel. Avoid injecting the solution too fast, since this may cause excessive tissue damage. Make sure not to inject any air.
12. Electroporate the fish immediately after the injection. Make sure that the fish is still under anesthesia. Keep the fish on the sponge and set the fish between tweezer-type electrodes, so that the electrodes are located on each side of the injection site. Do not press the electrode tweezers too tight but keep both electrodes in contact with the fish.
    1. Press the start button on the electroporator to give six 40 V, 50 ms pulses.
    2. Gently transfer the fish to the recovery tank.
    3. Clean the electrodes after each electroporation by swiping them with 70% ethanol.
       NOTE: Carefully monitor the well-being of the fish after the vaccination. Euthanize any fish showing signs of discomfort (a slow recovery from anesthesia, aberrant swimming, gasping) in 0.04% tricaine. After recovery, transfer the fish to the flow-through unit and feed it normally.