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DAPI/DA stain

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).

University of Wisconsin - Madison
Waisman Center Cytogenetics Lab