Materials

1. Culturing medium for larvae: 50 g Corn meal, 40 g ground raisins, 20 g sugar, 100 g baker's yeast, 8-10 g agar, 4 mL propionic acid, 1 L water.

2. Standard fly culture vials.

3. Saline solution for salivary gland dissections: 7.5 g NaCl, 0.35 g KCl, 0.21 g CaCl2, double-distilled or deionized water to 1000 mL.

4. 3H-Uridine (1 mCi/mL, specific activity 44 Ci/mM); for autoradiography.

5. Alcohol : acid fixative: 3 vol of 96% ethanol : 1 vol glacial acetic acid. Prepare fresh and keep at 4°C.

6. Formaldehyde fixative: 100 mM NaCl, 2 mM KCl, 10 mM NaH2PO4, pH 7.2-7.4, 2% formaldehyde. Prepare fresh.

7. Phosphate-buffered saline (PBS): 137 mM NaCl, 3 mM KCl, 8 mM NaH2PO4, 2 mM KH2PO4, pH 7.2.

9. 20X SSC (standard saline citrate): 3 M NaCl, 0.3 M Na-citrate, pH 7.0.

10. Denaturation solution: 70% Formamide in 2X SSC.

11. Hybridization buffer: 50% Deionized formamide, 1% dextran sulfate, in 2X SSC.

12. Blocking solution: 0.1% Bovine serum albumin (BSA) in PBST.

13. DIG DNA labeling kit, for incorporation of digoxygenin 11-dUTP (Roche).

14. Genius 1 buffer: 150 mM NaCl, 100 mM maleic acid (Roche).

15. Genius 2 buffer: Genius 1 buffer containing 0.5% Genius blocking reagent.

16. Antidigoxigenin-gold (0.8 nm) sheep IgG (Roche).

17. Silver enhancement reagents (Roche).

18. Gold (10 nm)-conjugated goat anti-rabbit IgG (Sigma-Aldrich). Dilute 1 : 50 in blocking solution following the manufacturer's recommendations.

19. Gold (10 nm)-conjugated goat anti-mouse IgG (Sigma-Aldrich). Dilute 1 : 50 in blocking solution.

20. Ilford L-4 emulsion (England) and Kodak D-19 developer solution.

21. Araldite M, Araldite M Hardener, and Araldite M Accelerator (Fluka).

22. Silicone solution (Serva).

23. 45% Acetic acid.

24. 2% Uranyl acetate in 70% ethanol (see Note 1).

26. Butanol.

27. Xylene.

29. Chloroform.

30. Siliconized microscopic slides (Superfrost slides, Erie Scientific Co.).

31. Siliconized cover slips (18 x 18 mm2).

33. Curved watchmaker's forceps, finely sharpened.

34. Needles for tissue dissections, stiff and very fine.

35. Diamond pencil.

36. Cups of aluminum foil or of thin plastic (see Note 2).

37. Objective marker (see Note 3).

38. Petri dishes.

39. Liquid nitrogen.

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