Encyclopedia of Experiments: Cancer Research
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Transcript
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.
