Distamycin A Plus DAPI Fluorescent Staining (Dst/DAPI)

In man, this technique produces brilliant fluorescence at the C-blocks of chromosomes 1, 9, and 16, at the heterochromatic part of the Y, and at a short arm segment of chromosome 15 (15pll).

Solutions:

1.. McIlvaine citric acid-Na2KP04 buffer, pH 7.0 (buffer A)

2. Distamycin A-HCl (Boehringer; Serva; Sigma) 0.2 mg/ml of buffer A

3. DAPl (4' - 6-Diamidino-2-phenylindole-2HCl') (Serva)
   Stock solution: 0.2 mg/ml of distilled water.
   Staining solutions: 0.2 - 0.4 µg/ml of buffer A


Staining method:

1. Flood slide with Dst-solution, cover with coverslip and
    incubate at room temp. for 5-15 mins.

2. Remove coverslip and rinse briefly with buffer, pH 7.0

3. Flood with DAPI solution, cover with coverslip and
    incubate in the dark at room temp. for 5-15 mins.

4. Rinse briefly with buffer A.

5. Mount in buffer A, remove excess buffer by blotting over coverslip.
   Seal with rubber solution.

Fluorescence microscopy:

excitation: approximately 360 nm

emission: approximately 460 nm

Note:

- Distamycin A tends to lack stability in aqueous solution unless frozen

- DAPI stock solution may be kept in the refrigerator for several
weeks without detectable deterioration or stored frozen.

- the stained preparations may exhibit rapid fading when first
examined but usually stabilize after a day or so stored at 4'C

Reference:

Schweizer, D..Ambros, P. and Andrle, M.: Modification of DAPI
banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A
Experimental Cell Research III, 327-332 (1978).

DAPI/Distamycin A Staining

Principle

The DAPI/Distamycin A staining technique is useful in identifying pericentromeric breakpoints in chromosomal rearrangements and in identifying chromosomes that are too small for standard banding techniques. Also, DAPI/DA is the method of choice for Yqh chromsome material in suspected Y autosome translocations.

Background

The DAPI/distamycin A fluorescent staining technique was first described by Schweizer, Ambros, and Andrle as a method for labeling a specific subset of C bands. (Gustashaw, 1991).

Stains

Distamycin A:

• Dissolve 2 mg distamycin A-HCl (Sigma) in 10 mL of McIlvaine's buffer, pH 7.0. Add a magnetic stirrer and place the foil-covered tube in a beaker of crushed ice. Stir for 15 to 30 minutes. Dispense in 1 mL aliquots and store at -20 degrees C for up to six months.

  DAPI
  

• Stock solution: Dissolve 2 mg DAPI-2 HCl (Sigma) in 10 mL of distilled water. Dispense in 1 mL aliquots, and store at -20 degrees C for up to six months.
  
• Working solution: Add 0.1 mL of stock solution to 100 mL of McIlvaine's buffer, pH 7.0. Store at 4 degrees C for up to six months.
  
• Buffer: McIlvaine's buffer, pH 7.0.
  
  Solution A: 0.1 mol/L citric acid (19.2g: q.s. to 1 liter with distilled water)
  
  Solution B: 0.2 mol/L Na2HPO4 (28.4g: q.s. to 1 liter with distilled water)
  
  The following formula can be used to prepare this buffer at various pHs:

  x mL A + (100 - x) mL B = 100 mL total volume

  For pH 4.1, x = 60
  
  For pH 5.5, x = 43.1
  
  For pH 7.0, x = 18.2
  

Procedure

1. Flood a slide with distamycin solution. Coverslip and incubate the slide, in the dark, at room temperature for 5 to 15 minutes.

2. Remove the coverslip and rinse briefly with pH 7.0 buffer.

3. Flood with DAPI working solution. Coverslip and incubate the slide, in the dark, at room temperature for 5 to 15 minutes.

4. Remove the coverslip and rinse the slide briefly with pH 7.0 buffer.

5. Observe with fluorescence (excitation: 340-380 nm; suppression: 430 nm).

6. Photograph as for other fluorescent techniques.

Special Note:
Use care when handling these substances. Wear gloves. Distamycin A tends to lack stability in aqueous solution unless it is frozen. DAPI stock solution can be frozen or refrigerated for several weeks without detectable deterioration. The stained preparations tend to fade rapidly, so photography should be immediate. Storing coverslipped slides at 4 degrees C for a day or so may stabilize the fluorescence.

References

Gustashaw, KM. Chromosome Stains. In The ACT Cytogenetics Laboratory Manual, Second Edition, edited by M. J. Barch. The Association of Cytogenetic Technologists, Raven Press, Ltd., New York, 1991.