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		Prep list  Drosophila 3rd instar larvae sterile petri dishes sterile forceps tungsten microneedles pulled Pasteur pipettes sterile glass pipettes paintbrushes pipettors depression slides sterile insect saline 70% and 95% ethanol Schneider's insect medium with 10% fetal calf serum ecdysone solution (1mg/mL in ethanol) Gentamicin reagent solution (50 mg/mL) Procedure 1. Use sterile technique throughout this experiment. 2. Prepare four 35-mm culture dishes by adding 2 mL of serum-supplemented Schneider's insect medium to each dish and 2 uL Gentamicin. a. To one plate, add 8 uL of ecdysone solution (1 mg powder per mL 95% ethanol). Final concentration of ecdysone 4 ug/ml. b. To the second plate, add 5 uL of ecdysone solution. c. Add 2 uL ecdysone solution to the third dish. d. The fourth control plate should receive 5 uL of 95% ethanol. 3. Obtain a large third-instar maggot from container (fig. 1, 2) and rinse it clean of debris by soaking it in several drops of saline on depression slide or other small container. Place container over ice to anesthetize and slow larvae responses. 4. Transfer larvae to several drops of cold saline solution on a clean glass plate (fig. 3). 5. Under a dissecting microscope remove as many imaginal discs as possible. a. Grasp the head of larva just behind the mouth parts with one pair of forceps. b. Grasp the middle of the larva with the other pair and gently pull the body away from the head. c. The imaginal discs will be concentrated in the anterior third of the body, attached to the tracheoles or gut (fig. 4). 6. Dissect the discs free of adhering tissue (fig. 5); the wings and legs discs are the largest and generally evert most drastically. 7. Dissect enough larvae to obtain 20 discs. 8. Transfer 4 discs into each dish using pulled Pasteur pipettes or tungsten microneedles. 9. Examine and photograph each disc. 10. Photograph and examine discs at two hours and 24 hours. 11. Allow discs to incubate at room temperature during development. Figure 1. Drosophila 3rd instar larvae in tube of fly medium. Figure 2. Drosophila 3rd instar larva (close-up).   Figure 3. Magnified photograph of 3rd instar Drosophila larva as viewed through dissecting microscope. Figure 4. Drosophila larva prior to removal of imaginal discs from larval body. Figure 5. Imaginal discs (im) free of debris and ready for transfer to culture dishes.