This article describes a method for delivering nephrotoxins into zebrafish embryos via intravenous microinjection. The procedure involves preparing a nephrotoxin solution, loading it into a microinjection needle, and carefully injecting it into the tail vein of the embryo.
Intravenous microinjection facilitates the delivery of molecules of interest into the bloodstream of a recipient animal. To deliver nephrotoxins into a zebrafish embryo, first, prepare an appropriate concentration of desired nephrotoxin solution.
Using a fine gel loading tip, load this nephrotoxin solution into a microinjection needle with a sharpened angular tip. Suspend the needle vertically to allow the nephrotoxin solution to fill the needle tip by gravity. Now, secure the loaded microinjection needle into the holder of a micromanipulator.
Subsequently, transfer an anesthetized zebrafish embryo into the prefabricated microinjection mold. Align the embryo in the mold with its head oriented within the well and the tail out of the well, improving the accessibility to the tail for microinjection. Next, precisely position the needle tip near the embryo.
Inject the nephrotoxin solution into the tail vein of the embryo. The injected nephrotoxic agents spread through the circulation and reach distant organs like the kidney. Gently retract the needle. Transfer the embryo into a fresh dish containing embryo media and allow it to recover. Incubate the embryo for a specific duration.
Within hours of injection, the nephrotoxins accumulate in the kidney and cause damage, leading to a decline in kidney function.
On the day of injection, prepare the desired nephrotoxin solution with the appropriate vehicle control. Vortex or mix it gently. Afterward, check the sides of the tube and top of the water column to ensure the drugs are dissolved in solution.
Load a trimmed microinjection needle with 2 to 3 microliters of nephrotoxin by threading a fine gel loading tip into the back of the needle and suspend the needle vertically with a piece of tape to allow the solution to fill the trimmed needle tip by gravity.
Once the needle tip is full, secure the loaded needle into the micromanipulator. Test the microinjection volume by placing a drop of mineral oil onto a micrometer slide and inject into the oil to evaluate the droplet size.
Next, remove the dish of embryos from the incubator and anesthetize them by adding 5 milliliters of 0.2% Tricaine to the embryo dish. Subsequently, transfer the embryos to the injection mold with a transfer pipette. Maneuver each embryo into a different well of the mold, placing the head in the deepest area of the well such that the trunk rests along the depression and the tail sticks out of it.
Now, insert the filled needle into the micromanipulator and position the needle tip next to an embryo. Gently insert the needle into the tail vein for microinjection.
The most critical step of the procedure is the microinjection. To ensure success, the needle must be cut at a sharp angle and must be moved carefully into position next to the embryo.
If the injection is successful, you should see the liquid entering the circulation. Co-injection of the nephrotoxicant with fluorescently conjugated dextran would enable the researcher to verify successful injection after the procedure. Afterward, gently remove the needle from the embryo.
Transfer the embryo to a clean dish and rinse to remove the Tricaine with fresh E3/PTU. Then, incubate the embryo to the desired time point to assess morphology, which can be documented by photography in methylcellulose mounting media, or process for experimental analysis as desired.
Note that recovery of embryos should be assessed every 12 to 24 hours after the injection to determine the percentage of individuals with edema, which commonly indicates renal injury.
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