3.3.1. Preparation of Squashes
2. Transfer a whole ovary to a fresh drop of Ephrussi-Beadle solution. Separate the ovarioles from peritoneal and tracheolated epithelial sheaths, mature eggs, and shelled oocytes, retaining NCs from terminal and subterminal egg chambers.
3. Using tiny needles, transfer five to six selected egg chambers to a drop of 45% acetic acid on a microscope slide. Incubate the egg chambers for a couple of minutes.
4. Cover the drop with a cover slip and make a squash as in Subheading 3.2., step 6.
5. Immerse the slide in liquid nitrogen and, when frozen, flip off the cover slip with a razor blade.
6. Give each slide three consecutive 5-min rinses in 96% ethanol.
7. Air-dry the slide.
8. Observe the chromosomes under phase-contrast optics (see Note 4).
3.3.2. Denaturation of Chromosomes
It is important here, and in Subheadings 3.3.3. and 3.3.4., to keep the slides
MOIST in all steps except where noted.
2. Wash the slides in 2X SSC at room temperature for 5 min.
3. Denature the slides in denaturation solution at room temperature for 1.5 min.
4. After denaturation, immediately transfer the slides to 70% ethanol at -20°C, and keep them there for 5 min.
5. Wash the slides in 80% and 96% ethanol at -20°C for 5 min each.
6. Air-dry the slides.
3.3.3. Hybridization With Probe
1. Mix a biotinylated DNA probe (0.1 |g per slide), competitor DNA (e.g., 0.2-0.3 |g salmon sperm DNA per slide) and water (see Note 5).
2. Heat the mixture at 100°C for 5 min.
3. Add 2 vol of 1.5X hybridization buffer and mix thoroughly.
4. Heat this mixture at 75°C for 2 min.
5. Drop 30 |L of the mixture per slide, cover with a cover slip, and seal the edges with rubber cement.
6. Keep the slides in a humid chamber with 2X SSC at 37°C overnight.
3.3.4. Detection of Signal
1. Wash the slides three times in 0.2X SSC at 60°C for 15 min.
2. Keep slides in blocking solution at 37°C for 30 min.
3. Drop 25 ||L of FITC-avidin per slide, cover with a cover slip, and incubate the slides in a humid chamber at 37°C for 30 min.
4. Wash the slides three times at 42°C for 5 min each with 4X SSC containing 0.1% Triton X-100. After these washes, the slides can be stored in 0.2X SSC at 4°C for at least 48 h.
5. Stain the slides with propidium iodide for 5-10 s or with DAPI for several minutes (see Note 6).
6. Air-dry the slides, add a drop of antifade reagent to an area of the squash, and cover with a cover slip.
7. View the chromosomes using a microscope appropriate for the DNA stain.
The brightness of weak fluorescent signals may be enhanced using the following procedures, one or more times. However, it should be kept in mind that all nonspecific signals will be enhanced as well.
1. Remove the cover slips from the slides to be treated.
2. Wash the slides twice at room temperature for 5 min with 4X SSC containing 0.1% Triton X-100.
3. Drop 25 |L of biotinylated antiavidin per slide, cover with a cover slip, and incubate the slides in a humid chamber at 37°C for 30 min.
4. Wash the slides twice in 4X SSC, 0.1% Triton X-100 at 42°C for 5 min.
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