Encyclopedia of Experiments: Biology
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- Prepare tapered injection needles by pulling thin-walled glass capillaries using a needle puller. Hold a needle under a microscope and break the tip, making sure that the opening is just large enough to pierce the adult fly cuticle without wounding it more than necessary. Measure the injection volume by loading your needle with a sterile dye and salt buffer solution and then ejecting it onto a drop of mineral oil on a stage micrometer. Use the scale on the micrometer to measure the diameter of the ejected droplet, which can be used to calculate the volume dispensed, and calibrate your injector settings accordingly.
Load the needle with your injection solution and mount it onto an injection nozzle. Anesthetize your flies with CO2 and arrange them on a CO2 fly pad that will keep them anesthetized. Once the flies are in the correct orientation for your experiment, proceed with the injections. In the example protocol, we will inject adult flies with fluorescein-labeled particles for in vivo imaging.
- After pulling thin-walled glass capillaries with a needle puller, use a micrometer to hold the needle under a microscope and use number five fine-point stainless steel tweezers to break the tip to a 100 micrometer tip diameter. To measure the volume of liquid that will be injected into each fly, load a capillary needle with sterile 5% food coloring in PBS and expel the liquid onto a drop of mineral oil on a 0.01 millimeter stage micrometer.
Dispense 10 microliters of 1.6 milligrams per milliliter particles onto a small square of parafilm and pull the liquid into the needle. Mount the needle into the injector nozzle and line the anesthetized flies along their designated area on the fly pad, ventral side up, with the heads oriented toward the front of the pad. Place the vials in corresponding areas on the bench and inject the flies at the upper corner of the abdomen with five 100 milliseconds pumps of liquid to deliver about 10 nanoliters of particles total. Transfer each fly into the appropriate vial as it is injected, noting the time on the vial.
Next, load a new needle with 0.4% Trypan Blue solution and set the pneumatic injector to 'gated' to allow a constant flow of air to push the liquid out of the needle. 30 minutes after the initial injection, inject each fly abdomen with Trypan Blue until the abdomens are full and distended. Mount the flies on microscope slides with electrical tape ventral side down, pushing the wings to the side of the fly to secure them to the tape. Then gently push the head into the tape to ensure that the fly will not move.