5 FISH on Blood or Bone Marrow Smears
93
Fluorescence in situ hybridization (FISH)
1. Add 100 III denaturation-buffer to the slides and cover with (24x50
mm) coverslips. [Denaturation-buffer: 70% (v/v) deionized formamide, 10% (v/v) mtered double distilled water, 10% (v/v) 20xSSC,
10% (v/v) phosphate buffer; make fresh as required; phosphate buffer:
prepare 0.5 M Na z HP0 4 and 0.5 M NaH z P0 4 , mix these two solutions
(1:1) to get pH 7.0, then aliquot and store at -20°C].
2. Incubate slides on a warming plate for 5 min at 75°C (see comment 4).
3. Remove the coverslips immediately with forceps and place slides in a
Coplin jar filled with 70% ethanol (4°C) to conserve target DNA as
single strands.
4. Dehydrate slides in ethanol (70%,90%, 100%, 4°C, 3 min each) and air
dry.
5. For each slide to be hybridized, treat the labeled YAC-, BAC- or cosmid-DNA according to the manufacturers instructions or add 1.5111 of
biotin or digoxigenin labeled chromosome specific satellite probes (e.g.
Oncor Inc., Vysis, Inc.) plus 0.6111 of Illg/1l1 COTl-DNA (Gibco) to 20
III of the hybridization-buffer in a 1.5-ml microtube, vortex and spin
down. (Hybridization-buffer: Dissolve 2 g dextran sulfate in 10 m150%
deionized formamide/2xSSC/50mM phosphate buffer for 3 h at 70°C.
Aliquot and store at -20°C).
6. Denature probe-solution at 75°C for 5 min and cool immediately on ice
to conserve probe DNA in single strands for satellite probes or handle
the probe according to the manufacturers instructions (e.g. do a prehybridization step at 37°C for 20 min for YACs, BACs, cosmids, Plclones).
7. Add 20 III of probe-solution onto each denatured slide, put 24 x 50 mm
coverslips on the drops and seal with rubber cement (Fixogum; Marabu).
8. Incubate slides for three nights at 37°C in a humid chamber (see comment 5).
9. Take the slides out of 37°C, remove rubber cement with forceps and the
coverslips by letting them swim off in 4xSSC/0.2%Tween (RT, 100-ml
Coplin jar) (see comment 6).
93
Fluorescence in situ hybridization (FISH)
1. Add 100 III denaturation-buffer to the slides and cover with (24x50
mm) coverslips. [Denaturation-buffer: 70% (v/v) deionized formamide, 10% (v/v) mtered double distilled water, 10% (v/v) 20xSSC,
10% (v/v) phosphate buffer; make fresh as required; phosphate buffer:
prepare 0.5 M Na z HP0 4 and 0.5 M NaH z P0 4 , mix these two solutions
(1:1) to get pH 7.0, then aliquot and store at -20°C].
2. Incubate slides on a warming plate for 5 min at 75°C (see comment 4).
3. Remove the coverslips immediately with forceps and place slides in a
Coplin jar filled with 70% ethanol (4°C) to conserve target DNA as
single strands.
4. Dehydrate slides in ethanol (70%,90%, 100%, 4°C, 3 min each) and air
dry.
5. For each slide to be hybridized, treat the labeled YAC-, BAC- or cosmid-DNA according to the manufacturers instructions or add 1.5111 of
biotin or digoxigenin labeled chromosome specific satellite probes (e.g.
Oncor Inc., Vysis, Inc.) plus 0.6111 of Illg/1l1 COTl-DNA (Gibco) to 20
III of the hybridization-buffer in a 1.5-ml microtube, vortex and spin
down. (Hybridization-buffer: Dissolve 2 g dextran sulfate in 10 m150%
deionized formamide/2xSSC/50mM phosphate buffer for 3 h at 70°C.
Aliquot and store at -20°C).
6. Denature probe-solution at 75°C for 5 min and cool immediately on ice
to conserve probe DNA in single strands for satellite probes or handle
the probe according to the manufacturers instructions (e.g. do a prehybridization step at 37°C for 20 min for YACs, BACs, cosmids, Plclones).
7. Add 20 III of probe-solution onto each denatured slide, put 24 x 50 mm
coverslips on the drops and seal with rubber cement (Fixogum; Marabu).
8. Incubate slides for three nights at 37°C in a humid chamber (see comment 5).
9. Take the slides out of 37°C, remove rubber cement with forceps and the
coverslips by letting them swim off in 4xSSC/0.2%Tween (RT, 100-ml
Coplin jar) (see comment 6).