An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

We aim to generate anthracycline-induced cardiotoxicity models in adult zebrafish so that we can discover new therapeutic genes and compounds. A reliable IP injection method is essential to ensure the success of this project. The previously developed IP injection methods are the classic and alternative techniques.

While the classic method penetrates the fish from the abdominal side through midline between the pelvic fins, the alternative method penetrates the fish from the dorsal side located between the pelvic and the anal fins. Owing to the toxicity of doxorubicin, significant body damage and mortality of the fish have been noted using the current two IP methods. Investigators need to be extensively trained before disease models can be reliably established in their hands.

We designed a new IP injection method by adjusting the needle penetration location and the needle penetration angle. We inject through a natural hole located in the ventral region between pectoral fins, and penetrate through a unique anterior to posterior direction. To begin, dissolve five milligrams doxorubicin powder in one milliliter of distilled water.

Aliquot the stock into 1.5 milliliter tubes. Next, group fishes with a body weight difference of less than 10%together. After anesthetizing the fishes in embryo water with tricaine, take the fish out of the water, dab both sides of the fish's body with clean filter paper to remove excess water.

Next, place the anesthetized fish in the groove of an embedded sponge with the abdomen facing upward. Fill a 10 microliter syringe equipped with a 34 gauge needle with five microliters of doxorubicin working solution. Insert the needle at a nearly zero degree angle from the midpoint of the pectoral fin towards the posterior side of the cardiac cavity.

Direct the needle towards the tail, and go beneath the silver skin. Slowly dispense the dock solution, then withdraw the needle along the original path to prevent leakage. The dock solution will color the abdomen red.