Injection of Adult Drosophila Flies: A Method of Compound or Label Delivery
Transcrição
– 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 para 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 para measure the diameter of the ejected droplet, which can be used para 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 de the correct orientation for your experiment, proceed with the injections. In the example protocol, we will inject adult flies with fluorescein-labeled particles for de vivo imaging.
– After pulling thin-walled glass capillaries with a needle puller, use a micrometer para hold the needle under a microscope and use number five fine-point stainless steel tweezers para break the tip para 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 de 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 de corresponding areas on the bench and inject the flies at the upper corner of the abdomen with five 100 milliseconds pumps of liquid para 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 para ‘gated’ para allow a constant flow of air para 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 para the side of the fly para secure them para the tape. Then gently push the head into the tape para ensure that the fly will not move.
