1. Fly stocks: All fly stocks were cultured at 16°C under uncrowded conditions on protein-rich medium (see item 2). All experiments were performed with females of either otu7/otu11, otu11/otu11, or otu11/otu11 carrying a Y chromosome (see Note 1). The X chromosome, bearing the otu11 allele carried the y, w, and sn3 mutations, whereas the chromosome with otu7 was free of markers. The FM3 chromosome was used to balance the stocks. Descriptions of all the markers and the FM3 balancer can be found in ref. 36.
The flies of the otu7/y w sn3 otu11 genotype were obtained by crossing FM3/ otu7 females with y w sn3 otu11/Y males. Therefore, the otu7 allele was always contributed by the mother and otu11 by the father. This point is stressed, because otu11/otu7 ovaries generate fewer functional oocytes (17).
2. Culturing medium: 100 g baker's yeast, 50 g corn meal, 40 g ground raisins, 20 g sugar, 10 g agar, 4 mL proprionic acid (serves as fungicide), made up to 1 L. Supplement with live moist yeast on surface.
3. Ephrussi-Beadle solution: 7.5 g NaCl, 0.35 g KCl, 0.28 g CaCl22H2O (or 0.42 g CaCl26H2O), and water to 1 L.
4. Leucobasic fuchsin: The methods for preparing this reagent can be found in Chapter 7.
5. Acetic orcein:
a. Pour 45 mL of 100% acetic acid into a flask, add 1 g of dry orcein. Cork the flask but NOT tightly!
b. Bring the acid with orcein to VERY WEAK boiling (several bubbles) for 25-30 s.
c. Add (carefully!) 55 mL of distilled water and heat back to a very weak boiling for 25-30 s.
d. Cool the flask to room temperature and add 1 N HCl in proportion 9 parts of staining solution to 1 part of HCl. Filter this mixture. This stock solution can be kept for months at room temperature.
e. Mix 2 parts of stock solution with 1 part of 45% acetic acid and filter this mixture. This staining solution is good for weeks.
6. Denaturation solution: 0.07 N NaOH, 2X SSC (see item 21).
7. Hybridization buffer (1.5X): 70% Formamide, 15% dextran sulfate, 3X SSC (see item 21).
8. Blocking solution: 4X SSC (see item 21), 0.1% Triton X-100, 2% Blocking Reagent (Roche Diagnostics GmBH, cat. no. 1 096 176) or 4% powdered milk (i.e., 4 g of powdered milk per 96 mL of blocking solution).
9. Cohen and Gotchell medium G: 25 mM di-sodium glycerophosphate, 10 mM KH2PO4, 30 mM KCl, 10 mM MgCl2, 3 mM CaCl2, 160 mM sucrose, 0.5% NP-40. This solution can be kept at 4°C for 2-3 d.
10. Formaldehyde fixative: 100 mM NaCl, 2 mM KCl, 20 mM sodium phosphate, 2% NP-40, 2% formaldehyde.
11. Phosphate-buffered saline (PBS)-glycerol: 33 mL of PBS (see item 24), 67 mL of glycerol.
12. TBS-Tween: 10 mM Tris-HCl, pH 7.15, 250 mM NaCl, 0.05% Tween-20 (see Note 2).
14. 45% Acetic acid.
15. 55% Lactic acid.
20. Water saturated with SO2.
21. SSC (0.2X, 2X, 3X, 4X): Stock solution 20X SSC contains 3 M NaCl and 0.3 M sodium citrate, pH 7.0. Other concentrations can be made by dilution of the stock solution.
24. PBS: 10X Buffer contains 11.5 g Na2HPO4, 2 g KH2PO4, 80 g NaCl, and 2g KCl per liter of water.
25. Fluoresein-isothiocyanate (FITC)-avidin dissolved in blocking solution (see item 8) at a final concentration of 25 |g/mL.
26. Biotinylated antiavidin antibodies (e.g., biotinylated, goat antiavidin D from Vector Laboratories, cat. no. BA-0300) dissolved in blocking solution (see item 8) at a final concentration of 1-10 |g/mL.
27. 4'-6-diamidino-2-phenylidole (DAPI) dissolved in 0.2X SSC (1 |g/mL).
28. Propidium iodide dissolved in 0.2X SSC (5 |g/mL).
29. Vectashield Antifade mounting medium.
30. Salmon sperm DNA.
31. Very fine needles (preferably tungsten), approx 0.2-0.3 mm in diameter, for dissection. Needles with curved tips are sometimes very helpful.
32. Blotting paper.
33. Microscope slides and cover slips.
34. Petri plates.
35. Liquid nitrogen.
3.1. Feulgen-Stained Ovarian Whole Mounts
2. Drop approx 20 ovaries into a small vial containing 1 N HCl at 60°C; maintain at this temperature for 14 min. To change solutions, gently pour out fluids. Use a clean micropipet to squirt in each new solution. Handle tissue gently.
3. Stain in leucobasic fuchsin until apical portions of the ovary take on a deep violet color (5-60 min).
4. Rinse ovaries two times in water saturated with SO2, 10% ethanol, 95% ethanol, 100% ethanol, ethanol-xylene (1 : 1), and xylene.
5. Transfer stained ovaries in a drop of xylene to a clean microscope slide. Tease the ovary into its constituent ovarioles. Put a drop of permount over the tissue and cover with a cover slip.
3.2. Orcein-Stained Squashes for Morphological Analysis of Chromosomes
1. To get females of the required genotype, put six to seven pairs of flies per vial with standard medium. Keep at 16°C. At this temperature, the life cycle of flies is approx 30 d.
2. Separate newly emerged females homozygous for the otu mutation and put them into vials with fresh medium. Keep the females at 16°C.
3. After 5-11 d, put etherized females in a Petri plate with Ephrussi-Beadle solution and dissect out the ovaries (see Note 1).
4. Gently transfer whole ovaries to acetic orcein for up to 1 h (see Note 2).
5. Transfer a stained ovary to a drop of 55% lactic acid on a microscope slide and then using needles separate the ovarioles from peritoneal and tracheolated epithelial sheaths, mature eggs and shelled oocytes, retaining NCs from terminal and subterminal normal stages 7-12 and p12s. Remove all unnecessary materials from the drop. The best NC chromosomes are from stages 9-11 and p12.
6. Cover the drop with a cover slip and spread the chromosomes by gently moving the cover slip back and forth (see Note 3). Put two to three layers of blotting paper on top of the cover slip and press down gently with your thumb. Remove the excess acid solution with blotting paper.
7. View the chromosomes under a phase-contrast microscope. These preparations remain suitable for analysis for 2-3 wk while stored at 4°C.
Was this article helpful?