CBG Banding

I. Preparation

1. Get out 4 clean coplin jars.
2. Label them as follows:
  • 0.2 N HCl
  • 1% BaOH)2
  • 2 X SSC
  • Leishmans stain
3. Prepare 0.2 N HC1 as follows:
  • 8 ml 1 N HCl
  • 32 ml distilled water
4. Prepare 1% Ba(OH)2 as follows:
  • 0.4 g Ba(OH)2
  • 40 ml distilled H20

5. Prepare 2 X SSC as follows: Pour 45-50 ml 2 X SSC into jar
6. Prepare Giemsa as follows:
  • 1 ml Leishmans stain
  • 3 ml Gurr buffer pH 6.8
 

II. Banding

1. Place slides in 0.2 N HCl for 1 hour.
2. Remove, rinse with distilled water.
3. Place slides in 1% Ba(OH)2 at 60° C for 15 seconds. Agitate the slide (BaOH2 will not go fully into solution.)
4. Remove, rinse with distilled water.
5. Place slides in 2 X SSC for 60° C for 1 hour.
6. Remove rinse with distilled water, allow to air dry. (Do not use air jet.)
7. Stain slides in Leishmans for about 2-4 minutes.
8. Remove, rinse with tap water.
9. Air dry.
10. Ready for analysis

III. Reagents

Barium hydroxide--Biochem Stores
Gurr's Improved R66 Giemsa--Biomedical Specialties
Hydrochloric Acid 1N--Biochem Stores
Leishmans stain ---Sigma.

 


C-Banding  (constitutive heterochromatin banding)

 

Constitutive heterochromatin includes repetative DNA, satellite DNA, and some non-repetative DNA and is found near the centromemeres and at the end of the long arm of the Y in human chromosomes. These regions stain darkly after treatment of the slide with acid, heat and alkyline solutions followed by staining with Leishman stain.


Notice the large heterochromatic regions on chromosomes 1 and 9.

 

C-Banding


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

Principle

To specifically stain the centromeric regions and other regions containing constitutive heterochromatin, i.e., the secondary contrictions of human chromsomes 1, 9, 16, and the distal segment of the Y chromosome long arm.

Equipment

    Circulating waterbath set at 65 degrees C;
    Four glass Coplini Jars with lids;
    9.0cm circles of Whatman #1 filter paper;
    Squirt bottle filled with distilled water;
    Slide warmer;
    Timer;

Reagents

Xylene substitute (Shandon #99900506) Toxic - avoid contact with skin and inhalation. Store in approved fire-proof cabinet. Discard in organic waste container.

Absolute methanal. (J.T. Baker, VWR #JT9070-3) Toxic - avoid contact with skin and inhalation. Store in approved fire-proof cabinet.

Glacial acetic acid. (J.T. Baker, VWR #JT9507-3) Toxic - avoid contact with skin and inhalation. Store in approved fire-proof cabinet.

Concentrated hydrochloric acid. (Fisher #A144-212) Toxic - avoid contact with skin and inhalation. Store in approved fire-proof cabinet.

Sodium chloride crystal. (Mallinckrodt, American Scientific Products #07581-1NY)

Sodium citrate crystal. (Mallinckrodt, American Scientific Products #0754-1NY)

Barium hydroxide crystal. (Mallinckrodt, American Scientific Products #3772-1NY)

3:1 Fixative: three parts absolute methanol with one part glacial acetic acid. Discard extra fixative down fum hood sink with copius amounts of water. Prepare fresh.

0.2 N HCl: add 4.15ml concentrated hydrochloric acid to 200ml of distilled water, mix and add water to 250ml. Stable 1 year.

2XSSC: dissolve 17.53g sodium chloride crystal and 8.82g sodium citrate crystal in one liter of distilled water. May be refrigerated in thightly capped bottle indefinitely.

2.5% BA(OH)2: dissolve 2.5g of barium hydroxide crystal in 100ml distilled water. This is a supersaturated solution. Store in thightly capped bottle at room temperature. Stable indefinitely.

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.