Fixation of Mitotic Chromosomes

1. Physiological solution: 0.7% NaCl in distilled water. Autoclave and store at 4°C.

2. Hypotonic solution: 0.5% Tri-sodium citrate dihydrate in distilled water. Autoclave and store at 4°C.

From: Methods in Molecular Biology, vol. 247: Drosophila Cytogenetics Protocols Edited by: D. S. Henderson © Humana Press Inc., Totowa, NJ

3. Fixative solution: Methanol/acetic acid/distilled water in the ratio 11 : 11 : 2 (v/v/v). Methanol is poisonous. It may be harmful by inhalation, ingestion, and skin absorption. Handle it in a chemical fume hood.

4. Liquid nitrogen. Caution: Liquid nitrogen is very dangerous because of its extreme temperature; wear cryo-mitts and a face mask.

5. Siliconized microscope slides.

8. Razor blade.

9. Absolute ethanol.

2.2. Orcein Staining

1. Physiological solution (see Subheading 2.1., item 1).

2. 1 mM Colchicine.

4. Hypotonic solution (see Subheading 2.1., item 2).

5. Fixative solution (see Subheading 2.1., item 3).

6. 2% Aceto-orcein solution: This solution is prepared by boiling synthetic orcein powder (Gurr) in 45% acetic acid for 45 min in a reflux condenser. To obtain good staining, it is important to remove the particulate matter from the aceto-orcein solution before use either by filtration through blotting paper or by cen-trifugation in a microcentrifuge.

7. Nail polish or depilatory wax (can be found in most cosmetic shops).

2.3. Giemsa Staining

1. 2% Giemsa in a phosphate buffer at pH 7.0. We routinely use Giemsa from Merck, but other Giemsa brands work just as well.

2. Euparal.

2.4. Hoechst Staining

1. Hoechst buffer (HB): 150 mM NaCl, 30 mM KCl, 10 mM Na2PO4, pH 7.0. Store at room temperature.

2. Hoechst solution: 0.5 pg/mL Hoechst 33258 dissolved in HB.

3. Mounting solution: 160 mM Na2HPO4, 40 mM sodium citrate, pH 7.0.

4. Rubber cement.

2.5. DAPI Staining

1. DAPI (4',6-diamidine-2-phenylindole dihydrochloride) stock solution: 100 pg/mL DAPI in distilled water. It can be stored in the dark at 4°C. It is a carcinogen and an irritant. It may be harmful by inhalation, ingestion, and skin absorption. Wear gloves and use it in a chemical fume hood.

2. 20X SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7.0. Sterilize by autoclaving and store at room temperature.

3. DAPI staining solution: 0.2 |g/mL DAPI dissolved in 2X SSC. Store at 4°C.

4. Vectashield-1000 (Vector Laboratories) or similar mounting medium containing glycerol and antifading compounds.

2.6. Quinacrine Staining

1. Absolute ethanol.

2. Quinacrine solution: 0.5% Quinacrine dihydrochloride (Gurr) dissolved in absolute ethanol.

3. Rubber cement.

2.7. N-Banding

1. 1 M Na2H2PO4 solution, pH 7.0, for heat treatment.

2. Giemsa solution: 4% Giemsa (Merck) in sodium phosphate buffer at pH 7.0.

3. Euparal or similar mounting medium.

3. Methods

3.1. Orcein Staining

1. Transfer third instar larvae into drops of approx 50 ||L of physiological solution placed on a siliconized slide and dissect the brains at room temperature (see Note 1).

2. Transfer the brains to a 35 x 10-mm covered Petri dish containing 2 mL of physiological solution and a drop of 1 mM colchicine and incubate for 1.5 h at 25°C (see Note 2).

3. Transfer the brains to a drop of hypotonic solution for 10 min at room temperature (see Note 2).

4. Transfer the brains to freshly prepared fixative solution for approx 20 s (check, however, the fixation under the dissecting microscope and continue to incubate the brains until they begin to become transparent).

5. Transfer four fixed brains individually into four small drops of 2% aceto-orcein placed on a very clean, dust-free nonsiliconized 20 x 20 or 22 x 22-mm2 cover slip and leave for 1-2 min.

6. Lower a very clean, dust-free slide onto the cover slip, which will adhere because of the surface tension. Invert the sandwich and squash between two or three sheets of blotting paper. To prevent the cover slip from sliding and the consequent damage to the preparation, squashing should be carried out in two steps. First, exert a gentle pressure to remove the excess of aceto-orcein and then squash very hard.

7. Seal the edges of the cover slip with either nail polish or melted depilatory wax (see Note 3) (see Fig. 1A-C for examples of orcein-stained chromosomes).

3.2. Fixation of Mitotic Chromosomes for Giemsa, Hoechst, DAPI, and Quinacrine Staining and N-Banding

1. Transfer third instar larvae into drops of approx 50 |L of physiological solution placed on a siliconized slide and dissect out the brains at room temperature (see Note 1).

Fig. 1. Examples of orcein- and DAPI-stained mitotic chromosome preparations of D. melanogaster neuroblasts. (A) Colchicine- and hypotonic-treated wild-type female metaphase; (B) colchicine- and hypotonic-treated female metaphase showing an autosomal symmetrical exchange (arrow); (C) colchicine- and hypotonic-treated mutant female metaphase showing a high degree of polyploidy; (D) DAPI-stained male metaphase; (E) cytological map of D. melanogaster heterochromatin. The diagrams are representative of prometaphase neuroblast chromosomes stained with Hoechst 33258 or DAPI. Only the heterochromatic portions of chromosomes are shown, with euchromatin depicted as a thin line. The entirely heterochromatic Y chromosome, the X chromosome, and the second, third, and the fourth chromosome heterochromatin are schematically represented from top to bottom. "C" indicates the position of the centromere; the location of the fourth chromosome centromere has not been precisely determined. Filled segments indicate bright fluorescence, cross-hatched segments indicate moderate fluorescence, hatched segments indicate dull fluorescence, and open segments indicate no fluorescence. These segments or bands are designated hi to h61, as indicated.

Fig. 1. Examples of orcein- and DAPI-stained mitotic chromosome preparations of D. melanogaster neuroblasts. (A) Colchicine- and hypotonic-treated wild-type female metaphase; (B) colchicine- and hypotonic-treated female metaphase showing an autosomal symmetrical exchange (arrow); (C) colchicine- and hypotonic-treated mutant female metaphase showing a high degree of polyploidy; (D) DAPI-stained male metaphase; (E) cytological map of D. melanogaster heterochromatin. The diagrams are representative of prometaphase neuroblast chromosomes stained with Hoechst 33258 or DAPI. Only the heterochromatic portions of chromosomes are shown, with euchromatin depicted as a thin line. The entirely heterochromatic Y chromosome, the X chromosome, and the second, third, and the fourth chromosome heterochromatin are schematically represented from top to bottom. "C" indicates the position of the centromere; the location of the fourth chromosome centromere has not been precisely determined. Filled segments indicate bright fluorescence, cross-hatched segments indicate moderate fluorescence, hatched segments indicate dull fluorescence, and open segments indicate no fluorescence. These segments or bands are designated hi to h61, as indicated.

2. Transfer the brains to a drop of hypotonic solution for 10 min at room temperature (see Note 2).

3. Transfer the brains to freshly prepared fixative solution for approx 20 s (check, however, the fixation under the dissecting microscope and keep the brains in the fixative until they begin to become transparent).

4. Transfer four fixed brains individually into four small drops of 45% acetic acid placed on a very clean, dust-free siliconized 20 x 20 or 22 x 22-mm2 cover slip and leave for 1-2 min.

5. Lower a very clean, dust-free slide onto the cover slip, which will adhere because of the surface tension. To prevent the cover slip from sliding and the consequent damage to the preparation, squashing should be carried out in two steps. First, exert a gentle pressure to remove excess acetic acid and then squash very hard.

6. Freeze the slide in liquid nitrogen.

7. Flip off the cover slip with a razor blade and immediately immerse the slide in absolute ethanol at room temperature. Air-dry the slides and store them at 4°C until further processing.

3.3. Giemsa Staining (see Note 4)

1. Rehydrate the air-dried slides in Hoechst buffer (HB) for 5 min at room temperature.

2. Stain in Giemsa staining solution for 10 min at room temperature (see Note 5).

3. Differentiate the chromosome preparations by washing the slides in tap water (see Note 6) and then air-dry the slides.

4. Mount in Euparal or similar medium.

3.4. Hoechst Staining (see Note 7)

1. Rehydrate the air-dried slides in Hoechst buffer (HB) for 5 min at room temperature.

2. Stain in Hoechst solution for 10 min at room temperature.

4. Air-dry the slides, keeping them in a vertical position.

5. Mount either in HB or mounting solution.

6. Seal around the edges of the cover slip with rubber cement.

7. To reduce fluorescence fading, store the slides in the dark at 4°C for 1-2 d before observation (see Note 8).

3.5. DAPI Staining

1. Rehydrate the slides for 5 min in 2X SSC at room temperature.

2. Stain in DAPI solution for 5 min at room temperature.

3. Wash briefly (approx 5 s) in 2X SSC solution.

4. Air-dry the slides, keeping them in a vertical position.

5. Mount in Vectashield-1000 or in a similar medium containing glycerol and antifading compounds. For examples of DAPI-stained chromosomes, see Fig. 1D,E.

3.6. Quinacrine Staining (see Note 9)

1. Immerse the slides with fixed chromosomes in absolute ethanol for 5 min.

2. Stain the slides in quinacrine solution for 10 min.

3. Wash the slides twice (5 s each wash) in absolute ethanol.

4. Air-dry the slides, keeping them in a vertical position.

5. Mount in distilled water.

6. Seal around the edges of the cover slip with rubber cement.

7. Store the slides in the dark at 4°C before observation to reduce fluorescence fading and to improve the degree of differentiation (see Note 8).

1. Immerse the slides with fixed chromosomes in 1 M Na2H2PO4, pH 7.0, at 85°C, and incubate for 15 min.

2. Transfer the slides to distilled water at room temperature.

3. Stain the slides in Giemsa solution for 20 min at room temperature.

4. Differentiate the chromosome preparations by washing the slides in tap water (see Note 6).

5. Air-dry the slides.

6. Mount in Euparal or similar medium.

7. Examine the slides using phase-contrast optics.

4. Notes

1. Use two fine forceps (e.g., Dumont no. 5 Biologie) to easily dissect the brains. The larval mouth parts and the posterior part of the larval body should be grasped and then pulled apart. Because the brain usually remains attached to the head together with several imaginal discs and the salivary glands, the more rigid mouth parts should be completely removed with the forceps.

2. Colchicine incubation followed by hypotonic treatment permits one to obtain a large number of good metaphase figures (200-400 per brain) that can be analyzed for chromosome morphology, presence of chromosome aberrations and degree of ploidy. However, to examine the degree of chromosome condensation, the colchicine must be omitted because this substance disrupts spindle microtu-bules and induces metaphase arrest and overcontraction of chromosomes. Hypotonic treatment improves metaphase chromosome spreading and causes sister-chromatid separation, allowing examination of chromosome condensation. Because hypotonic shock disrupts anaphase (10), these figures are almost always absent in hypotonically treated brains. To observe all phases of mitosis and to evaluate the mitotic index and frequency of anaphases, the dissected brains should be squashed in aceto-orcein without colchicine treatment and hypotonic shock, although in such preparations, chromosome morphology is not so well defined (11).

3. Well-sealed slides can be stored for 1-2 mo at 4°C without substantial deterioration.

4. Giemsa staining is required for permanent preparations. This can be done by staining fixed chromosomes.

5. The timing of Giemsa staining varies with the Giemsa brand and should be adjusted to obtain the desired staining.

6. Because Giemsa stain is additive, if the chromosomes are not sufficiently stained, the slide can be stained again in 2% Giemsa until the desired staining is obtained.

7. This protocol (5,12) is a modified version of the protocol described by Latt (13) for mammalian chromosomes. Other protocols have been described (14,15).

8. Fluorescence fading depends on both the lamp and the filter sets used for epifluorescence. If the degree of fading is too high, the slides may be mounted in Vectashield-1000 or in a similar medium containing glycerol and antifading compounds.

9. This quinacrine staining protocol was developed by Gatti et al. (5). Other quina-crine banding techniques have been described by Vosa (16), Ellison and Barr (17) and Faccio Dolfini (18).

10. The N-banding procedure is essentially that of Funaki et al. (19) with minor modifications (7). Best results are obtained when fixed preparations are aged for 2-5 d at 4°C before processing.

References

1. Gatti, M. and Pimpinelli, S. (1992) Functional elements in Drosophila melanogaster heterochromatin. Annu. Rev. Genet. 26, 239-275.

2. Ashburner, M. (1989) Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

3. Gonzalez, C. and Glover, D. M. (1993) Techniques for studying mitosis in Drosophila, in The Cell Cycle: A Practical Approach (Fantes, P. and Brooks, R., eds.), IRL, Oxford, pp. 143-175.

4. Gatti, M., Tanzarella, C., and Olivieri, G. (1974) Analysis of the chromosome aberrations induced by X-rays in somatic cells of Drosophila melanogaster. Genetics 77, 701-719.

5. Gatti, M., Pimpinelli, S., and Santini, G. (1976) Characterization of Drosophila heterochromatin. Staining and decondensation with Hoechst 33258 and quina-crine. Chromosoma 57, 351-375.

6. Pimpinelli, S., Pignone, D., Gatti, M., and Olivieri, G. (1976) X-ray induction of chromatid interchanges in somatic cells of Drosophila melanogaster: variation through the cell cycle of the pattern of rejoining. Mutat. Res. 35, 101-110.

7. Pimpinelli, S., Santini, G., and Gatti, M. (1976) Characterization of Drosophila heterochromatin. II. C- and N-banding. Chromosoma 57, 377-386.

8. Bonaccorsi, S., Santini, G., Gatti, M., Pimpinelli, S., and Coluzzi, M. (1980) Intra-specific polymorphism of sex chromosome heterochromatin in two species of the Anopheles gambiae complex. Chromosoma 76, 57-64.

9. Bonaccorsi, S., Pimpinelli, S., and Gatti, M. (1981) Cytological dissection of sex chromosome heterochromatin of Drosophila hydei. Chromosoma 84, 391-403.

10. Brinkley, B. R., Cox, S. M., and Pepper, D. A. (1980) Structure of the mitotic apparatus and chromosomes after hypotonic treatment of mammalian cells in vitro. Cytogenet. Cell Genet. 26, 165-176.

11. Gatti, M. and Baker, B. S. (1989) Genes controlling essential cell cycle functions in Drosophila melanogaster. Genes Dev. 3, 438-453.

12. Gatti, M. and Pimpinelli, S. (1983) Cytological and genetic analysis of the Y chromosome of Drosophila melanogaster. I. Organization of the fertility factors. Chromosoma 88, 349-373.

13. Latt, S. A. (1973) Microfluorimetric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc. Natl. Acad. Sci. USA 70, 3395-3399.

14. Holmquist, G. (1975) Hoechst 33258 fluorescent staining of Drosophila chromosomes. Chromosoma 49, 333-356.

15. Hazelrigg, T., Fornili, P., and Kaufman, T. C. (1982) A cytogenetic analysis of X-ray induced male steriles on the Y chromosome of Drosophila melanogaster. Chromosoma 87, 535-559.

16. Vosa, C. G. (1970) The discriminating fluorescence patterns of the chromosomes of Drosophila melanogaster. Chromosoma 31, 446-451.

17. Ellison, J. R. and Barr, H. J. (1971) Differences in the quinacrine staining of the chromosomes of a pair of sibling species: Drosophila melanogaster and Drosophila simulans. Chromosoma 44, 424-435.

18. Faccio Dolfini, S. (1974) The distribution of repetitive DNA in the chromosomes of cultured cells of Drosophila melanogaster. Chromosoma 44, 383-391.

19. Funaki, K., Matsui, S., and Sasaki, M. (1975) Location of nucleolar organizers in animal and plant chromosomes by means of an improved N-banding technique. Chromosoma 49, 357-370.

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