We have tested this protocol using antibodies against Mod (30) and HP1 (Mal'ceva, N. I. and Demakova, O. V., unpublished data; Koryakov, D. E., unpublished data). An example of this staining is shown in Fig. 8. This method is based on protocol 30 in ref. 37 and ref. 38, with minor changes. It is important AT ALL STEPS of this protocol, except the last, to KEEP THE SQUASHES WET!
3.4.1. Preparation of Squashes
All solutions for this method should be kept at 4°C during all procedures.
1. Follow Subheading 3.2., steps 1-2.
2. Dissect whole ovaries in a drop of Cohen and Gotchell medium G (containing NP-40) or in Ephrussi-Beadle solution containing 0.5% NP-40 at room temperature and transfer them to a fresh drop of the same solution. Incubate ovaries in this solution for 3-5 min.
3. Transfer the ovaries to formaldehyde fixative for 10-20 min (see Note 7).
4. Transfer the ovaries to a drop of 45% acetic acid on a microscope slide and keep there for 2-3 min.
5. Using fine needles, separate ovarioles from peritoneal and tracheolated epithelial sheaths, mature eggs and shelled oocytes, retaining NCs from terminal and subterminal chambers (stages 7-12). Remove all unnecessary tissue material from the drop.
6. Cover with a cover slip and make a squash (see Subheading 3.2., step 6).
7. Immerse the slide in liquid nitrogen until frozen and flip off the cover slip with a razor blade.
9. Store the slides in TBS at 4°C. If the slides are not to be used within 24 h, then store in PBS-glycerol at -20°C. Slides can be kept in this solution for 1-2 d, and even up to a week.
3.4.2. Staining With Antibodies
1. Remove the slides from storage medium (PBS-glycerol) and wash three times in TBS-Tween at room temperature, 5 min each wash.
2. Remove the slides from TBS-Tween, dry the bottom of the slides quickly with a piece of blotting paper, and add 15-20 |L of primary antibodies to the squash immediately, then cover it with a cover slip (see Note 8).
3. Keep the slides in a humid chamber at 4°C overnight (see Note 9).
4. Rinse the slides three times, for 5 min each, with ice-cooled (4°C) TBS-Tween.
5. Remove the slides from TBS-Tween, dry the bottom of the slides quickly with a piece of blotting paper, and immediately add 15-20 |L of secondary FITC-labeled antibodies to the squash, then cover with a cover slip.
6. Keep the slides in a humid chamber at room temperature for 2 h.
7. Rinse the slides three times for 5 min each in TBS-Tween at room temperature.
8. Air-dry the slides.
9. Stain slides with DAPI and view with ultraviolet (UV) light under a fluorescence microscope.
1. Ovarian function is extremely sensitive to nutritional and environmental resources. Presence of males accelerates ovary development and egg maturation (6). An additional Y chromosome also influences the rate of ovary development and the cytological quality of NC chromosomes. Ovaries of Y-containing females were observed to develop faster and more of their egg chambers reached stage 10 during 4-6 d at 16°C. The percentage of polytene chromosomes of good quality in NCs of Y-containing females is higher. However, polytene chromosomes of the best quality were obtained in a stock homozygous for otu11 and carrying Dp(l;l)pn2B.
2. Staining times vary widely. We used from 30 min at room temperature to overnight at 4°C and did not find noticeable differences. As NC chromosomes are more diffuse than SG chromosomes, they are stained weakly and long treatments by acetic orcein may intensify staining. At the same time, cytoplasm is also saturated by orcein, which seriously hampers chromosome exploration.
3. The drop must be large enough for the cover slip to move freely, or chromosomes will be destroyed. If the drop is too large, nuclei will be washed out with excess liquid.
4. During this step, a majority of slides may be rejected, because we use only chromosomes of such length (see Subheading 1.3.1.) and having banding patterns that can be easily identified with chromosome maps. Unfortunately, these "good" slides constitute no more than 25-30% of all slides made. Others contain too much cytoplasm, which hides chromosomes. Some chromosomes cannot be identified because they look like "luminous cords" without banding patterns. In comparison with SG squashes, where each slide contains dozens of good nuclei, in NC squashes we select slides that contain as few as one to two good nuclei per slide. Putting more than 8-10 egg chambers on a slide adds too much cytoplasm, which prevents chromosome observation.
5. Volumes of components of this mixture depend on the number of slides and the component concentrations. For example, for one slide, you need 1 ||L of biotinylated probe at a concentration of 0.1 |g/|L, 2-3 |L of competitor DNA at 0.1 |g/|L, and 7-8 |L of water (i.e., to bring the final volume to 10 |L). To this mixture add 20 |L of hybridization buffer.
6. Duration of staining can vary. You can decrease the concentrations of staining solutions and simultaneously increase the duration of staining to obtain the best results.
7. Time of fixation can be varied widely. It is important for SG chromosomes not to fix them for too long, because this can dramatically decrease chromosome quality. We believe that NC chromosomes must be fixed longer. We fixed ovaries for 10 min in the case of HP1 and 15-20 min in the case of Mod.
8. Protocol 30 in ref. 37 states that the volume of antibodies should be 40 |L. However, if you use a cover slip and rubber cement, 15-20 |L of antibodies are enough.
9. You can incubate the slides with primary antibodies for 2-4 h at room temperature, but we find it is better to incubate the slides overnight at a lower temperature (4-10°C).
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