Preparation and Immunostaining of Polytene Chromosome Squashes (PROT01)

Christine James, Paul Badenhorst & Bryan Turner
Institute of Biomedical Research
Institute of Biomedical Research
University of Birmingham Medical School
Birmingham
B15 2TT, UK

email feedback to: c.h.james.1@bham.ac.uk

Last reviewed: 27 Jan 2005 by Cristina Chioda, Becker lab, München and Ina Dahlsveen, Exiqon (formerly of Becker Lab, München).

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Introduction

The polytene chromosomes found in the salivary glands of Drosophila larvae (and other diptera), provide a valuable model system in which microscopical techniques can be used to study the functioning of the interphase genome. Chromosome banding patterns, revealed by simple DNA stains or phase contrast, provide markers by which individual chromosomes can be identified and by which specific genes or genomic regions can be located. Immunostaining provides an additional level of resolution by allowing non-histone proteins or modified histones to be located to such genomic regions, or associated with specific chromatin functions (eg. transcription) or chromatin types (e.g. heterochromatin).

However, procedures that give the best polytene chromosome preparations (squashes) with the most clearly-resolved bands, involve treatment with concentrated acetic acid. If unmodified, such procedures result in extraction of all, or nearly all, the histones and most non-histone proteins. To prevent this, it is necessary to pre-fix the chromosomes with formaldehyde. Unfortunately, such fixation compromises the spreading of polytene chromosomes. A squashing procedure that allows successful immunolabelling requires a fine balance between fixation that is sufficient to retain a high enough proportion of the protein of interest, while not preventing the preparation of suitably spread and banded chromosomes. We have found the following procedure to be successful for squashing and immunolabelling with several different antisera to modified histones H3 and H4.

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Procedure

Preparation of Third Instar Larva

1. Drosophila melanogaster strain w¹¹¹⁸ was maintained in polystyrene vials (Fisher cat #DRO-105-010W) on Yeast-Dextrose-Agar culture medium, supplemented with additional active yeast (Fermex Five Star) at room temperature (22°C) (see comment 1);
2. Adult flies were allowed to lay eggs for 2-3 days, to the point where the larvae would hatch under uncrowded conditions (to allow optimum growth) and were then transferred to fresh vials;
3. "Wandering" third instar larvae were used for the preparation of polytene chromosome squashes ie. larvae that have migrated out of the media up the walls of the tube but have not yet formed pupae (see comment 2) .

Polytene Chromosome Squashes

1. Third instar larvae were dissected in solution 1 using two sets of fine forceps (Jeweler's Dumont No.5 forceps, Sigma cat #F6521). One pair of forceps is used to grip the body of the larva while the mouth-hooks are grasped firmly with the other pair. The forceps are slowly drawn apart to separate the mouth-hooks from the rest of the body. The salivary glands should follow the mouth hooks with little other tissue attached. However, white fat body tissue may remain attached to the salivary glands and as much as possible should removed before squashing;
2. As soon as the gland had been extracted, it was placed into a well of a 96-well microtitre plate (flexible, round-bottomed) containing 250µl of solution 2;
3. After 30-60 seconds, solution 2 was removed by pipette and replaced by 250µl of solution 3 (see comment 3);
4. The glands were left in solution 3 for 2 minutes before being removed by pipette (glands and solution 3 sucked up together in a total volume of 20µl) and placed onto a non-siliconised glass coverslip;
5. The coverslip was picked up onto a poly-L-lysine coated slide (Sigma), tapped in each corner ~4 times and a couple of times lightly on top of the actual gland. Using the lead end of a pencil, a zigzag line was traced from the bottom left of the slide to the top right. This movement forces solution 3 to move out of the coverslip at one end, which is supposed to aid good spreading. Following this, hard downward thumb pressure was applied to the pair of glands in the same direction as the movement of the zigzag line and of solution 3 (see comment 4);
6. The slide was labelled so as to mark the position of the coverslip, and hence of the gland. This procedure was repeated 4 times so that 4 slides were ready for examination. The 4 preparations were examined under phase contrast to check the quality of the spreads. In our hands, approximately 1 in 4 slides have good spreads of a quality suitable for immunolabelling;
7. The good slides were frozen in liquid nitrogen, and the coverslip flicked off with a razor blade;
8. The slides were then immersed in 80% ethanol and stored at -20°C until use. We have never stored slides for more than a few days before labelling (see comment 5).

Labelling Polytene Chromosome Squashes

1. The slides were taken out of 80% ethanol and placed in 40% ethanol for 10 minutes;
2. The slides were immersed in PBS for 10 minutes;
3. The slides were placed in blocking solution (1% BSA (see comment 6), 0.5% Triton X100 in PBS) for 1 hour;
4. Slides were removed and 50µl of primary antibody diluted in blocking solution (1% BSA in KCM) was placed directly onto the squashed pair of glands (see comment 7). A small rectangle of Parafilm was placed over the antibody to prevent evaporation (see comment 8);
5. The slides were placed in a humid chamber for 1 hour at 4°C (see comment 9);
6. The slides were washed in KCM for 15 minutes;
7. Slides were labelled with 50µl of secondary antibody (see comment 10) (using the same procedure as for primary antibody labelling) for 1 hour (see comment 7);
8. Slides were washed in KCM for 15 minutes;
9. Slides were removed and mounted in Vectashield (Vecta Laboratories) containing DAPI (1µg/ml) as a DNA counter-stain. The coverslip was sealed with nail varnish before viewing under the microscope.

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Materials & Reagents

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Author Notes

1. A typical example of a "good" immunolabelled chromosome squash is shown in Figure 1.
2. Various procedures for preparing and immunolabelling polytene chromosomes have been published, mostly based on the work of Sally Elgin and colleagues nearly 30 years ago (Paro, 2000 and references therein). Trial and error may be required to work out the exact protocol that works in your own laboratory with your own fly stocks. The protocol described here differs in significant detail from the one we used in our previous laboratory with a different source of flies (Turner et al., 1992). Results with the old protocol have been unsatisfactory in the new environment.

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Reviewer Comments

Reviewed by: Cristina Chioda, Becker lab, München and Ina Dahlsveen, Exiqon (formerly of Becker Lab, München).

1. 18°C works well in our hands.
2. Larvae that are about to pupate are also too old and can be distinguished by having everted spiracles.
3. Thirty seconds is best.
4. Tapping lightly with the back of a brush (or similar) starting in the middle of the squash and moving out in concentric circles also produces good spreads. It is important that there is enough liquid left under the coverslip for it to move slightly during tapping.
5. After freezing slides can be immersed in PBS directly and stored for some hours at room temperature, or can be stored in PBS, 3% BSA, 0.1% Triton at 4°C for up to one week. After immersion in PBS, slides can also be stored in methanol at -20°C for at least two weeks, and can be transferred directly into PBS (without re-hydration) before staining.
6. 0.7% NaCl OK
7. Quality of squashes decreases the older solution 2 is, therefore making fresh solution 2 every 30 minutes could increase number of good squashes obtained. Small aliquots of 3.7% paraformaldehyde in PBS and 7.4% paraformaldehyde in water stored at -20°C are useful in this case. Aliquots are thawed just before use to make up solutions 2 and 3 respectively.
8. Some antibodies may require higher BSA concentrations, i.e. 3%.
9. Normal blocking solution (PBS, 1-3% BSA, 0.1% triton) can be used for antibody dilution.
10. 20 µl of solution is enough when the staining is performed under a coverslip. Place 20µl of antibody solution on a coverslip and pick up with the slide.
11. Some antibodies need o/n incubation at 4°C.
12. Washing after primary antibody could be extended. We typically do 3x5 minutes in PBS, 2x15 minutes in PBS 1-3% BSA, 0.1% triton.

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Figures

Figure 1. Polytene chromosome spread prepared as described in the protocol and immunostained with antiserum against H3 tri-methylated at lysine 4 (H3K4me3). The centric heterochromatin at the chromocentre is unstained and there is banded staining along the chromosome arms.

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References

1. Paro, R. Mapping protein distribution on polytene chromosomes by immunostaining. In, Drosophila Protocols, eds. W. Sullivan, M. Ashburner, R. Scott Hawley, Cold Spring Harbor Press, New York, 2000.
2. Turner, B.M., Birley, A.J. and Lavender, J. (1992). Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei. Cell, 69: 375-384.

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Xin Tong :

Posted 585 days ago

this is really nice protocol to me , but I have a problem: how to get rid of the debris?Thanks

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