This method was successfully used in the Cytogenetics Laboratory of the Laboratory of Pathology in Seattle, Washington, USA.

Principle

Chromosomes are treated with quinicrine mustard solution, a fluorescent stain, to identify specific chromosomes and structural rearrangements. It is expecially useful for distinguishing the Y chromosome (also Y bodies in interphase nuclei) and various polymorphisms involving satellites and centromeres of specific chromosomes.

Equipment

Mettler analytical balance;
3 one-liter bottles;
8 coplin jars;
10ml serological pipette;
50ml beaker;
clean coverslip;

Reagents

Quinicrine mustard dihydrochloride, 25mg. (Sigma #Q2000) Store desiccated in light-free container at -20 degree C.

Dibasic sodium phosphate, reagent grade. (J.T. Baker, VWR #JT3828-1)

Citric acid, anhydrous reagent grade. (J.T. Baker, VWR #JT0122-1)

Sucrose, reagent grade. (J.T. Baker, VWR #JT4072-1)

Quinicrine mustard (QM) solution: dissolve 2mg of QM in 10ml distilled water and dilute to 40ml with McIlvances buffer with a final concentration of 50mcg/ml. Store in coplin jar (light-free) for 1-2 weeks in refrigerator.

McIlvanes buffer (pH 7.0)

a. Stock solution A: dissolve 28.38g of dibasic sodium phosphate in 900ml of distilled water; then add water up to one liter. Store in tightly capped bottle in refrigerator.

b. Stock solution B: dissolve 21.01g of citric acid in 900ml of distilled water; then add water up to one liter. Store the same as solution A.

c. McIlvanes buffer working solution: to make 200ml of solution at pH 7.0, combine 164.7ml of A with 35.3ml of B. Mix well. Store in refrigerator in thightly capped bottle.

Sucrose syrup: dissolve with heat 6g of sucrose in 10ml McIlvanes buffer (pH 7.0), then store in tightly capped bottle in refrigerator.

Procedure

1. G-band slides to facilitate identification of chromosomes. Photograph 2-3 metaphase spreads for documentation and for comparison after C-banding.

2. Remove oil from G-banded slide thoroughly with at least two rinses of fresh Xylene substitute.

3. Destain slide by dipping in 3:1 fix; wipe bottom of slide, place on 40-60 degree C slide warming tray until beads of solution are formed: then blot gently with bibulous paper. These four steps should be repeated until beads are clear.

4. Place dry, destained slide in 0.2 N HCL for one hour. After 1/2 hour turn on pre-set waterbath and start to filter BA(OH)2 through #1 Whatman filter paper into Coplin jar.

5. Rinse slide (treated in 0.2 N HCl) in Coplin jar filled with distilled water.

6. Place rinsed slide in freshly filtered BA(OH)2 solution for two minutes.

7. Rinse with distilled water in squirt bottle (some force is required to remove BA(OH)2 crystals).

8. Place rinsed slide in Coplin jar (in waterbath) filled with 2XSSC at approximately 62.5 degrees C for one hour.

9. Remove slide slowly and rinse gently in Coplin jar filled with freshly distilled water.

10. After drying, the slide should be stained as follows:
- For peripheral blood specimen, stain 90 seconds with 1:5 Wright's stain (For specific information about stain preparation, see G-banding procedure).

References

Sumner, A. A simple method for demonstrating centromeric heterochromatin. Exp. Cell Res. 75:304-306, 1972.