14

Chromosome Staining and Banding Techniques

Kevin A. Hahn and Penny K. Riggs

1. Introduction Each chromosome in the somatic-cell complement can be uniquely identified

by followmg a number of different banding procedures. The banding patterns are highly characteristic. The International System for Cytogenetic Nomenclature (ISCN) provides schematic representations, or Ideograms, of human chromo- somes correspondmg to approx 400, 550, and 850 bands per haploid set (I). Although under constant revision, its principles rest on a numbering system based on major bands as they appear from the centromere outward along each chromo- some arm. Similar standards have been established for other mammalian spe- cies, and recent literature should be reviewed for appropriate standards and revisions before attempting to karyotype a metaphase specimen.

To the cytogeneticist, the appearance of well-prepared, clearly banded chro- mosomes has an aesthetic appeal that is often difficult for the noncytogeneti- cist to comprehend. In part, this may be attributable to steps in some procedures that have no obvious scientific explanation but that nevertheless do materially affect results. Many published staining methods devised m one laboratory require modification m another laboratory. Despite these somewhat mystical aspects of the craft, rigid adherence to times, concentrations, temperatures, and pH can result m methods that are highly reproducible and reliable (2).

The finding of a chromosome abnormality does not always imply multiple defects in morphogenests, growth disturbances, and mental retardation in a patient. Some anomalies cause no harm. Their effects on the phenotype obvi- ously depend on both the quality and quantity of the genetic material mvolved.

The methods described m this chapter represent procedures that the authors have found useful m their laboratories. Optimal use of the microscope and good photographic procedures are essential.

From Methods m Molecular Me&one, Vol 14 Tumor Marker Protocols E&ted by M Hanausek and 2 Walaszek 0 Humana Press Inc , Totowa, NJ

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240 Hahn and Riggs

2. Materials 2.1. Slide Preparation

1 Absolute methanol 2. Deionized or distilled water. 3. Microscope slides. 4 Nonsterile 2-4-mL Pasteur pipets.

2.2. Solid Staining

1. 0 025M Phosphate buffer (pH 6 8) 0.025M KHzP04 (3 4 g/L) titrated to pH 6 8 with 50% NaOH. Make fresh as required

2. 10% Gremsa stam. 5 mL of Gremsa (Gurrs) plus 45 mL of 0 025Mphosphate buffer (pH 6 8). Make fresh as required

2.3. Giemsa Banding (G-Bands)

1. 0.025M Phosphate buffer (pH 6.8). 0.025M KH2P04 (3 4 g/L) titrated to pH 6.8 with 50% NaOH. Make fresh as required.

2. Detomzed or disttlled water 3. 10% Hydrogen peroxtde: 33 mL 30% Hz02 with 67 mL distilled or deionized

water. Maintained at 4C Make fresh as reqmred 4 0.025% Trypsm (Grand Island Biologic Company, Grand Island, NY)* 5 mL of

0 25% trypsm to 45 mL of 0.025M phosphate buffer, pH 6 8. Maintain at 4C Thus solutron must be used immediately or replaced after 30-60 min of use

5. 0 02% Fetal bovine serum (FBS). 1 mL serum added to 50 mL phosphate buffer (pH 6 8), maintained at 4C Make fresh as required

6. 10% Giemsa stain 5 mL of Giemsa (Gurrs) plus 45 mL of 0 025M phosphate buffer (pH 6.8) Make fresh as required.

2.4. Reverse Banding (R-Bands)

1. Sorensens buffer, solution A: 0.5M KH2P04 (6.8 g/100 mL deionized or dis- tilled water). Stable at room temperature for 1 mo

2. Sorensens buffer, solution B: 0.5M Na2HP04 (7.1 g/100 mL deionized or distilled water). Stable at room temperature for 1 mo.

3. Sorensens buffer (pH 6 8): 3 1 4 mL of Sorensens buffer solution A, 22 8 mL of Sorensens buffer solution B, 945 8 mL deionized or distilled water Stable at room temperature for I mo

4 Sorensens buffer (pH 8.0). 2.8 mL of Sorensens buffer solution A, 32 4 mL of Sorensens buffer solution B, 964.8 mL deronized or distilled water. Stable at room temperature for 1 mo.

5. Hoechst 33258 (Sigma, St. LOUIS, MO): 1 mg Hoechst m 1 L Sorensens buffer (pH 6.8) Make fresh as required.

6. 2X SSC: 0.3M NaCI, 0.03M trtsodmm citrate. Make fresh as required 7 3% Giemsa stain 3 mL Gurrs Geimsa m 97 mL Sorensens, pH 8.0 Make fresh

as required.

241 Stainmg and Bandmg

3. Methods 3.1. Slide Preparation (see Note 1)

1. Soak new mrcroscope slides m absolute methanol overnight. 2 Rinse shdes three times m deromzed water 3 Shdes can be stored m water and used wet or dry depending on preference. 4 Centrifuge the cell suspenston containing metaphase chromosomes (see Chapter

13 m this volume) at 1 OOg for 10 mm 5 Discard all but l-2 mL of the supernatant 6. Gently resuspend the cell pellet mto a fine cell suspension in the remainmg

supernatant usmg the tip of a Pasteur pipet 7. Aspirate a small amount of cell suspenston mto a Pasteur prpet and expel about

three drops carefully m three different posrtrons on each slide. 8 Place the slide at a 45 angle and let the slide an-dry Spreading 1s achieved by

the movement of the periphery of the drop outward until an-dried

3.2. Solid Staining (see Note 2)

1 Place an-dried slides m the Gremsa stam for 8 mm 2 Rinse the slides twice m deionized or drsttlled water 3. An-dry 4 Mount, rf necessary, with a cover slip

3.3. Giemsa Banding (G-Bands) (see Note 3) Dry the slides on a 60C warmmg tray or incubator for at least 4 h prior to staining Immerse the slide into a 10% hydrogen peroxide solution for 15 s, rinse m detomzed or distilled water and dram slide well (shake off excess water) Cytoplasm that may cover metaphase chromosomes will be removed by this procedure and permit better exposure of the chromatm to the trypsm treat- ment (2) This will result m more consistent staining of the slides prepared from different samples Immerse the slide mto the trypsm solutton for about 10-15 s. This time will vary consrderably depending on the quantity of sample on the slide and the actrvrty of the trypsm. Therefore, use test slides to determine optimal time of trypsin expo- sure and concentratron (2) Immerse the slide 5-7 times in FBS solution (serum m the media contains ar-anti- trypsm to arrest the drgestron process) Longer treatment at thus step may adversely affect banding (2). Rinse the slide with phosphate buffer Place the slide m Gremsa stain for about 8-10 mm. Time may vary. Rinse the slide with phosphate buffer. Rinse the slide with deromzed or dtstrlled water

9. Allow slide to an-dry m a vertical position 10 Mount, if necessary, with a cover shp.

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3.4. Reverse Banding (R-Bands) (see Note 4)

Hahn and Riggs

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2 3 4

5 6

7 8 9

10

11

Dry slides for at least 1 wk at room temperature or dry overnight on a 60C slide warmer. Immerse the shdes m Hoechst solution for 30 mm at room temperature (3,4) Add fresh Hoechst solution to slide and cover with cover slip Illuminate the slides under uv light for 30 mm The uv lamp should be 2 5 cm from the slide (3,4) Rinse the slides m 2X SSC Incubate for 60-90 mm m 2X SSC at 65C Tap occasionally to dislodge bubbles (3,4) Rinse the slides m Sorensens phosphate buffer, pH 8 0 Stain with 3% Glemsa stam for 10 mm Rinse the slides three times m Sorensens buffer, pH 8 0, and twice m drs- tilled water Air-dry slides at room temperature for 30 mm and then on a 50C slide warmer for 1 h Mount, if necessary, with a cover slip

4. Notes 1 Laboratories vary m their preparation of microscope shdes Some use shdes

straight from the manufacturers box, whereas others soak slides m alcohol, fixa- tive, ether, or chromic acid, and dry and polish slides prior to use Some use a detergent to remove all traces of grease; however, the detergent may also leave a coating layer on the shde Whether pretreated for extra cleanhness or not, shdes should be clean and grease-free to ensure good spreadmg of chromosomes There are many varlatlons of the spreadmg method described in Subheading 3.1. The quality of spreading may be influenced by temperature; high temperatures may cause overspreading of chromosomes and cell breakage, whereas low tempera- tures may mhlblt spreading. This 1s caused, m part, by the different rates of evapo- ration of the fixative (3). Addltlonally, chromosome spreading quality may be improved by varying the height from which the cell suspension 1s dropped onto the slide. Sohd-stain a representative shde (Subheading 3.2.) and observe for metaphase cells. If protem-stained debris obscures the visualization of chromo- somes, recentrlfuge the cell suspension, discard all but 1 mL of the supernatant, resuspend the cells m fresh fixative, let stand for 10 mm at room temperature, centrifuge, discard all but 1 mL of the supernatant, and make another shde Once condltlons are appropriate (I e , metaphase chromosomes with mmlmal overlap and crisp sohd-stained chromosomes), make a mmlmum of 10 nonstamed slides for chromosome banding The cell pellet can then be mamtamed for 4-6 wk m a sealed centrifuge tube kept under refrigeration

2. Stammg procedures that provide a uniform unbanded appearance to chromo- somes are referred to as solid or conventional staining. Although banded chro- mosome studies are far more informative, solid-stained preparations can be useful for studies on chromosome breakage since scoring gaps and breaks can be dlffi-

Staining and Banding 243

cult in lightly stained chromosomes. Slides can be destained by soaking in Carnoys fixative (three parts absolute methanol and one part glacial acetic acid) and subsequently stained by another technique.

3 Giemsa banding (G-banding) has become the most widely used technique for the routine staining of mammalian chromosomes The most usual methods to obtain this staining are to treat the slides with a protease, such as trypsm, or incubate the slides in hot saline-citrate, although a variety of other methods have been used The quality of banding is greatly influenced by the trypsmization procedure (2). Shdes should be momtored as they are prepared since it may be necessary to vary the length of trypsm exposure or Giemsa staining time

4 Bands that are negative, which appear pale by G-banding, stain darkly by R-band- mg. Conversely, dark positive G-bands appear pale using R-banding techniques R-banding can be achieved by mcubatron in hot salme solution followed by Giemsa staining. Although the pattern of staining appears to reflect the structural and functtonal composition of chromosomes, the chemical basis for the stammg reactions remams obscure (3,4)

References 1. Hamden, D. G and Klmger, H P (1985) ISCN Znternatzonal System for Human

Cytogenetzc Nomenclature Karger & Basel, New York, pp l-l 17. 2 Hahn, K. A., Richardson, R C , Hahn, E. A., and Chrisman, C. L (1994) The

diagnostic and prognostic importance of cytogenetic aberrations identified in spontaneously occurrmg canme malignant lymphoma Vet Path 31,528-540.

3 Ronne, M. (1989) Chromosome preparation and high resolution banding tech- niques: a review. J Dairy Scz 72, 1363-1377.

4. Droum, R., Lemieux, N , and Richer, C. L. (1988) High-resolution R-banding at the 1250-band level technical considerations on cell synchronizatton and R-bandmg (RHG and RBG) Cytobios 56, 107-125