1. Two no. 5 forceps (Fine Science Tools), one should be Biologie (extrafine tips).

2. Insect medium, such as Schneider's medium (Sigma-Aldrich).

3. 20% Bovine serum albumin (BSA) (Sigma-Aldrich), on ice.

4. 10X Phosphate-buffered saline (PBS): 80 g NaCl, 2 g KCl, 2 g KH2PO4, pH 7.3, on ice.

5. Fixative: 4% Formaldehyde fixative made fresh from paraformaldehyde (see Note 1). Dissolve 0.65 g Na2PO4 in 80 mL of double-distilled water in a flask. Add 4 g of paraformaldehyde (Polysciences), cap the flask loosely, and heat to 60-70°C in a fume hood. Stir until the paraformaldehyde has dissolved, cool, add 0.4 g NaH2PO4, dissolve, then add double-distilled water to 100 mL and filter through a 22-|m filter. Adjust to pH 7.4 if necessary. This solution is not stable and should be made fresh (see Notes 2 and 3).

6. Wash solution: 1X PBS, 1% BSA, 0.1% Triton X-100 (Fisher Biotech, electrophoresis grade), 0.03% sodium azide (see Notes 3 and 4).

7. Block-permeabilization solution: 1X PBS, 1% BSA, 0.3% Triton X-100, 0.03% sodium azide (i.e., wash solution with extra Triton X-100) (see Notes 3 and 4).

8. 100X DAPI stock: 10 mg DAPI/100 mL methanol. Store in the dark at -20°C (see Note 3).

9. Final clearing solution: 1X PBS, 0.03% Brij35 (Sigma-Aldrich), 0.03% sodium azide (see Note 3).

10. Slides and cover slips of the appropriate thickness for the microscope to be used.

11. Vectashield mounting medium (Vector Laboratories).

12. Clear nail polish.

3. Methods

3.1. Dissection (see Note 5)

1. Select a third instar larva. Third instar larvae are large, wandering larvae that have anterior spiracles with papillae. The anterior spiracles are located on either side of the black mouth hooks and, in third instar larvae, the fingerlike papillae make them look like hands. (First instar larvae have no papillae.) See Fig. 1.

2. In cold PBS, hold the larva with one forceps at about mid-body, and pull the head off with the other forceps. How you grasp the head determines which discs you get. Grasp the mouth hooks and spiracles to pull out all discs (except the genital disc). The wing, haltere, and T3 leg disc are usually associated to the main tracheal trunk, which resembles a white cord. The remaining disc complexes are with the mouth hooks and associated structures and may also come with the salivary glands, brain, and part of the gut. If you grasp just the mouth hooks and pull abruptly, you can get only the eye-antennal discs attached to the mouth hooks. Figure 1 shows the approximate locations of the discs, and the groupings that are likely to be together.

3. Carefully pull away the salivary glands, fat bodies, brain, gut, and excess material if any is attached to the discs (see Note 6).

4. Transfer the discs to a 0.5-mL Eppendorf tube containing 500 |L of PBS on ice. Ten microliters of 20% BSA added to the PBS will keep the discs from sticking to the Eppendorf tube.

3.2. Fixation

1. Remove the PBS from the disc complexes by aspirating with a syringe or vacuum device and add 500 |L of fixative. Fix for 20 min at room temperature (RT).

2. Wash three times with the wash solution for 10 min each wash. Fixed tissue can be stored for only a short time at 4°C in aqueous solution (see Note 7).

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