Intrathoracic Injection: Compound Delivery to the Adult Zebrafish Heart

- To begin, place a wet sponge with a slit on a Petri dish. Next, anaesthetize an adult fish with tricaine solution and carefully transfer it into the slit with the ventral side facing up. Place the Petri dish under a stereo microscope and observe the movement of the beating heart under the skin.

Position a mincroinjection needle at a 45 degree angle from the body axis above the beating heart located between the operculum and the pectoral girdle. Gently, advance the needle through the skin into the pericardium, a thin membrane that surrounds the heart. Deliver the injection mix into the pericardium.

The shot contains a tracer dye such as phenol red to track the solutions movement. Withdraw the needle from the thorax. Transfer the fish immediately into the recovery tank containing system water and monitor it for complete recovery from anesthesia. In the following protocol, we will perform an intrathoracic injection to deliver a test compound and examine its effects on heart regeneration.

- Use a net to catch an adult fish and transfer it into the anesthetic solution. After one to two minutes, when the fish stops swimming and the movement of operculum is reduced, touch the fish with a plastic spoon to make sure it does not react to any contact. Next, quickly and carefully transfer the fish into the groove of the wet sponge with the ventral side up. The head of the fish should point away from the operator's dominant hand.

Under the stereo microscope, carefully observe the movement of the beating heart under the skin of the fish. Visually, determine the injection point above the beating heart and in the middle of the triangle defined by the ventral cartilaginous plates. Insert the tip of the capillary at an angle between 30 and 45 degrees relative to the body axis. Gently penetrate the skin with the tip of the microcapillary into the pericardium.

Once the needle is inside the pericardium, press the pedal of the microinjector device to complete the injection. After injection, gently withdraw the capillary from the thorax, and immediately transfer the fish into a tank with system water for recovery. Monitor the fish until total recovery from anesthesia. To analyze the effects of injection, collect the heart at the desired time point and prepare it for further analysis.