Chromosomal Staining.pptx

Each chromosome in the somatic-cell complement can be uniquely identified by following 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 chromosomes corresponding 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 chromosome arm.
To the cytogeneticist, the appearance of well-prepared, clearly banded chromosomes has an aesthetic appeal that is often difficult for the non-cytogeneticist to comprehend.

1. Chromosomal Staining

2. INTRODUCTION Each chromosome in the somatic-cell complement can be uniquely identified by following 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 chromosomes corresponding 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 chromosome arm. To the cytogeneticist, the appearance of well-prepared, clearly banded chromosomes has an aesthetic appeal that is often difficult for the non-cytogeneticist to comprehend.

3. 2. Materials 2.1. Slide Preparation 1 Absolute methanol 2. Deionized or distilled water. 3. Microscope slides. 4 Nonsterile 2-4-mL Pasteur pipets.

4. Solid Staining 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. 10% Giemsa stain. 5 mL of Giemsa ( Gurr’s ) plus 45 mL of 0.025M phosphate buffer (pH 6.8). Make fresh as required

5. 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. Deionized or distilled water 3. 10% Hydrogen peroxide: 33 mL 30% H2O2 with 67 mL distilled or deionized water. Maintained at 4°C Make fresh as required 4. 0.025% Trypsin (Grand Island Biologic Company, Grand Island, NY)* 5 mL of 0.25% trypsin to 45 mL of 0.025M phosphate buffer, pH 6.8. Maintain at 4°C Thus solution 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 4’C Make fresh as required 6. 10% Giemsa stain 5 mL of Giemsa ( Gurr’s ) plus 45 mL of 0.025M phosphate buffer (pH 6.8) Make fresh as required.

6. 2.4. Reverse Banding (R-Bands) 1. Sorensen’s buffer, solution A: 0.5M KH2P04 (6.8 g/100 mL deionized or distilled water). Stable at room temperature for 1 month 2. Sorensen’s buffer, solution B: 0.5M Na2HP04 (7.1 g/100 mL deionized or distilled water). Stable at room temperature for 1 mo. 3. Sorensen’s buffer (pH 6.8): 31.4 mL of Sorensen’s buffer solution A, 22.8 mL of Sorensen’s buffer solution B, 945 8 mL deionized or distilled water Stable at room temperature for I month 4 Sorensen’s buffer (pH 8.0). 2.8 mL of Sorensen’s buffer solution A, 32 4 mL of Sorensen’s buffer solution B, 964.8 mL deionized or distilled water. Stable at room temperature for 1 month. 5. Hoechst 33258 (Sigma, St. LOUIS, MO): 1mg Hoechst in 1 L Sorensen’s buffer (pH 6.8) Make fresh as required. 6. 2X SSC: 0.3M NaCI , 0.03M trisodium citrate. Make fresh as required 7 3% Giemsa stain 3 mL Gurr’s Geimsa m 97 mL Sorensen’s, pH 8.0 Make fresh as required.

7. 3.1. Slide Preparation 1. Soak new microscope slides in absolute methanol overnight. 2 Rinse slides three times in deionized water 3 Slides can be stored in water and used wet or dry depending on preference. 4 Centrifuge the cell suspension containing metaphase chromosomes at 1OO g for 10 min 5 Discard all but l-2 mL of the supernatant 6. Gently resuspend the cell pellet into a fine cell suspension in the remaining supernatant using the tip of a Pasteur pipet 7. Aspirate a small amount of cell suspension into a Pasteur pipet and expel about three drops carefully in three different positions on each slide. 8 Place the slide at a 45” angle and let the slide an-dry Spreading is achieved by the movement of the periphery of the drop outward until air-dried

8. Solid Staining (see Note 2) 1 Place air-dried slides in the Giemsa stain for 8 minutes 2 Rinse the slides twice m deionized or distilled water 3. Air-dry 4 Mount, if necessary, with a cover slip

9. Giemsa Banding (G-Bands) (see Note 3) 1. Dry the slides on a 60°C warming tray or incubator for at least 4 h prior to staining 2. Immerse the slide into a 10% hydrogen peroxide solution for 15 s, rinse in deionized or distilled water and drain slide well (shake off excess water). Cytoplasm that may cover metaphase chromosomes will be removed by this procedure and permit better exposure of the chromatin to the trypsin treatment (2) This will result m more consistent staining of the slides prepared from different samples 3. Immerse the slide in to the trypsin solution for about 10-15 s. This time will vary considerably depending on the quantity of sample on the slide and the activity of the trypsin. Therefore, use test slides to determine optimal time of trypsin exposure and concentration (2) 4. Immerse the slide 5-7 times in FBS solution (serum in the media contains alpha-antitrypsin to arrest the digestion process) Longer treatment at thus step may adversely affect banding (2). 5. Rinse the slide with phosphate buffer 6. Place the slide in Giemsa stain for about 8-10 mm. Time may vary. 7. Rinse the slide with phosphate buffer. 8. Rinse the slide with deionized or distilled water 9. Allow slide to an-dry in a vertical position 10 Mount, if necessary, with a cover slip.

10. Reverse Banding (R-Bands) 1. Dry slides for at least 1wk at room temperature or dry overnight on a 60°C slide warmer. 2. Immerse the slides in Hoechst solution for 30 min at room temperature (3,4) 3. Add fresh Hoechst solution to slide and cover with cover slip 4. Illuminate the slides under UV light for 30 min The UV lamp should be 2.5 cm from the slide (3,4) 5. Rinse the slides m 2X SSC 6. Incubate for 60-90 min in 2X SSC at 65°C Tap occasionally to dislodge bubbles (3,4) 7. Rinse the slides m Sorensen’s phosphate buffer, pH 8.0 8. Stain with 3% Giemsa stain for 10 mm 9. Rinse the slides three times m Sorensen’s buffer, pH 8 0, and twice in distilled water 10. Air-dry slides at room temperature for 30 min and then on a 50°C slide warmer for 1 h 11. Mount, if necessary, with a cover slip

11. 4. Notes 1 Laboratories vary in their preparation of microscope slides. Some use slides straight from the manufacturer’s box, whereas others soak slides in alcohol, fixative, 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 slide Whether pretreated for extra cleanliness or not, slides should be clean and grease-free to ensure good spreading of chromosomes There are many variations of the spreading method described in Subheading 3.1.

12. The quality of spreading may be influenced by temperature; high temperatures may cause overspreading of chromosomes and cell breakage, whereas low temperatures may inhibit spreading. This is caused, in part, by the different rates of evaporation of the fixative (3). Additionally, chromosome spreading quality may be improved by varying the height from which the cell suspension is dropped onto the slide. Solid-stain a representative slide (Subheading 3.2.) and observe for metaphase cells.

13. If protein-stained debris obscures the visualization of chromosomes, re-centrifuge the cell suspension, discard all but 1 mL of the supernatant, resuspend the cells in fresh fixative, let stand for 10 min at room temperature, centrifuge, discard all but 1mL of the supernatant, and make another slide. Once conditions are appropriate (I e , metaphase chromosomes with minimal overlap and crisp solid-stained chromosomes), make a minimum of 10 non-stained slides for chromosome banding. The cell pellet can then be maintained for 4-6 wk m a sealed centrifuge tube kept under refrigeration

14. 2 . Staining procedures that provide a uniform in-banded appearance to chromosomes are referred to as solid or conventional staining. Although banded chromosome studies are far more informative, solid-stained preparations can be useful for studies on chromosome breakage since scoring gaps and breaks can be difficult in lightly stained chromosomes. Slides can be destained by soaking in Carnoy’s fixative (three parts absolute methanol and one part glacial acetic acid) and subsequently stained by another technique.

15. 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 trypsin, 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 trypsinization procedure (2). Slides should be monitored as they are prepared since it may be necessary to vary the length of trypsin exposure or Giemsa staining time 4 Bands that are negative, which appear pale by G-banding, stain darkly by R-banding. Conversely, dark positive G-bands appear pale using R-banding techniques R-banding can be achieved by incubation in hot saline solution followed by Giemsa staining. Although the pattern of staining appears to reflect the structural and functional composition of chromosomes, the chemical basis for the staining reactions remains obscure (3,4)