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Procedure: A. Dissection of imaginal discs 1. Place a vial of wildtype larvae into an ice bucket to anesthetize the larvae. 2. Add 3 to 4 drops of Insect Ringer's Solution onto a depression slide. 3. Use a spatula to obtain a wild-type third instar larva. Third instar larvae are white and can be found crawling along the wall of the vial, rather than burrowed in the food medium. 4. Place the larva on the depression slide and observe it under the dissecting microscope. 5. Using two fine forceps, hold the head and the tail of the larva and gently pull. The larval body will split one-third from the anterior end. This is ideal because most of the imaginal discs are located in the anterior region of the larva. 6. Discard the posterior portion of the larva. Look for the imaginal discs. These should look like a bunch of grapes attached to the central nervous system. Some discs might have detached. Look for these discs in the debris. Consult the figure above (Cruz. Fig. 17.2) if you are not sure what imaginal discs look like. *Footage of imaginal disc dissection
B. Staining imaginal discs for lacZ expression 1. Pre-warm assay buffer in a 37°C water bath. 2. Place the imaginal discs onto a new depression slide containing 3 to 4 drops of Insect Ringer's Solution. 3. After 5 minutes, transfer the imaginal discs onto a new depression slide containing 3 to 4 drops of the chilled fixative. Keep the Eppendorf tube containing the fixative in the ice bucket when not in use. 4. Wait for 10 minutes to allow the fixative to take effect. 5. Transfer the imaginal discs to a new depression slide and rinse with 3 to 4 drops of Insect Ringer's solution. Repeat to make sure all fixative is washed off. 6. Transfer the imaginal discs to a new depression slide containing 3 to 4 drops X-gal assay buffer. 7. Transfer 600mL of the assay buffer to a new vial and add 4.5mL of 20% X-gal in DMF. This will be your staining solution. If you see a white precipitate in the vial, place the vial in a 37°C water bath until all the precipitate dissolves; the solution should be clear. 8. Transfer the imaginal discs onto a depression slide containing 3 to 4 drops of the staining solution. Allow 15 minute exposure. LacZ expression pattern will appear blue. 9. Transfer the imaginal discs onto a clean depression slide containing 3 to 4 drops of Insect Ringer's solution. 10. Observe the imaginal discs under the dissecting microscope. Look for lacZ staining pattern. 11. Photograph the imaginal discs using the Olympus SZx12 Stereomicroscope with DP12 digital camera. 12. Repeat step A and B with En-lacZ third instar larva. *Protocol provided by Judith Cebra-Thomas, Swarthmore College
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