Materials Used
4’,6’-Diamidino-2-phenylindole dihydrochloride (DAPI ) - Fisher Scientific Cat# AC20271-0100
Triton X-100 - Fisher Scientific Cat# BP151-100
Glycerol - Fisher Scientific Cat# G31-1
Paraformaldehyde (16% w/v aqueous solution) - Fisher Scientific Cat# AA433689M
9-well spot plate - Fisher Scientific Cat# 13-748B
Dissecting needle - Fisher Scientific Cat# S97398
Jewelers Forceps - Fisher Scientific Cat# 12-460-110
Slides - Fisher Scientific Cat# S95352
Cover slips (18 x 18 mm) - Genesee Scientific Cat# 29-115
Clear Nail Polish - Genesee Scientific Cat# 88-128
Electrical Tape
1X Phosphate Buffered Saline (PBS): 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4
PPE Required: gloves, lab coat, safety goggles
Protocol
1. Add 3-4 drops of PBS-TX (1X PBS + 0.1% Triton X-100) into 1 well of a 9-wellspot plate.
2. Transfer larvae to the well containing PBS-TX
3. Dissect larvae in PBS-TX by pulling the larva in half, inverting the head section and removing the trachea and fat, leaving the imaginal discs.
Leave eye discs attached to mouth hooks and brain
Discard the posterior portion of the larva
Some of the discs might be found in the debris
4. Transfer the discs to a 1.5 mL Eppendorf tube containing chilled fixative (4% formaldehyde in PBS-TX) and incubate on ice for 20 minutes.
5. Remove fixative solution and discard in appropriate waste container.
6. Add 500 μL of PBS-TX. Incubate at RT for 5 minutes.
7. Remove PBS-TX and discard in appropriate waste container.
8. Add 1mLof PBS-TX + 1 μL DAPI [1mg/mL stock solution]. Incubate at RT for 5 minutes.
9. Remove PBS-TX/DAPI and discard in appropriate waste container.
10. Add 500 μL of PBS-TX. Incubate at RT for 5 minutes.
11. Use a dissecting needle and forceps to dissect imaginal discs away from mouth hooks and brain
12. Prepare slide using electrical tape to bridge for coverslip.
13. Add 90 μL 50% glycerol to slide [v:v with 1X PBS].
14. Cut the tip off a P200 tip to allow discs to enter tip.
15. Transfer discs with P200 tip to prepared slide.
16. Cover with coverslip and seal with clear nail polish. View using fluorescence microscope
Figure 1. Schematic representation of late third-instar Drosophila larvae, showing the approximate locations and shapes of the imaginal discs. (Reprinted from Bodenstein 1950). Note that most of the imaginal discs are located in the anterior region of the larva.
Figure 2. Larval dissection. (A) Grasping third-instar larva with forceps. (B) Isolated CNS-eye-antennal disc complex. (ad) Antennal disc; (br) brain; (ed) eye disc; (mh) mouth hooks; (os) optic stalk; (vnc) ventral nerve cord.