Zebrafish Cardiomyopathy Model: Drug-Induced Cardiotoxicity

1. DOX Injection in Adult Fish

1. Place a clean 100 mm Petri dish with a sponge inside it, under a dissection microscope, then adjust the focus. Cut the sponge to make a cavity of about 4 cm in length to hold a fish. Make a longer cavity for a larger fish.
2. Prepare a 34 G needle with a 10 µL micro-syringe. Rinse the needle with 1x HBSS buffer to remove any bubbles and blocks from the syringe and tubing.
3. Anesthetize the adult fish in embryo water containing 0.16 mg/mL tricaine for 2 min.
   NOTE: Prolonged anesthetization over 5 min followed by DOX injection can easily cause fish death.
4. Soak the sponge in embryo water with tricaine, and transfer the fish onto the sponge for injection.
5. Perform IP DOX injection by either of the two methods described below.
   1. Classic IP injection
      1. Position the fish with the abdomen up in the cavity of the sponge. Quickly insert the needle, with a 45° angle to the fish body into the midline between the pelvic fins, and penetrate approximately 1-2 mm. Release all DOX solution slowly. Wait 5 s before pulling out the needle. Check the DOX delivery by a visible red color in the fish belly.
   2. Alternative IP injection
      1. Place the fish laterally onto the sponge with the anterior to the right. Gently stabilize the fish using a blunt end forceps with the left hand, and hold the micro-syringe with the right hand.
      2. Position the needle below the lateral line above the pelvic fin, with the bevel facing up. Pointing at the 7 o'clock position at a 45° angle, insert the needle 3-4 mm to the fish cavity located between the pelvic and the anal fins, and then slowly depress the plunger. Check the DOX delivery by a visible red color in the fish belly.
6. Quickly transfer the injected fish to a clean crossing tank filled with fresh system water to allow the fish to recover. Rinse the needle once with 1x HBSS buffer between injections.