3 FISH on Chromosome Preparations of Peripheral Blood
77
9. Formalin-buffer is replaced by 100 mIl xPBS for 2 min (RT, with gentle agitation).
10. Finally, slides are dehydrated by an ethanol series (70%, 90%, 100%,
3 min each) and air dried (see comment 6).
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) filtered 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
0:0 to get pH 7.0, then aliquot and store at -20°C).
2. Incubate slides on a warming plate for 2-3 min at 75°C (see comment 7).
3. Remove the coverslips immediately with forceps and place slides in a
Coplin jar fllied 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 DNA-probes according to manufacturers instructions or add 1.0 III of biotin or digoxigenine-Iabeled chromosome specific satellite probes (e.g. Oncor Inc., Vysis, Inc.) plus 0.6 III of IIlg!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 ml50% deionized
formamide/2xSSC/50mM phosphate buffer for 3 h at 70°C. Aliquot
and store at -20°C} (see comment 8).
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 manufactures instructions (e.g. do a prehybridization step at 37°C for 20 min for any kind of library, YACs, BACs,
cosmids or PI-clones).
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 9).
77
9. Formalin-buffer is replaced by 100 mIl xPBS for 2 min (RT, with gentle agitation).
10. Finally, slides are dehydrated by an ethanol series (70%, 90%, 100%,
3 min each) and air dried (see comment 6).
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) filtered 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
0:0 to get pH 7.0, then aliquot and store at -20°C).
2. Incubate slides on a warming plate for 2-3 min at 75°C (see comment 7).
3. Remove the coverslips immediately with forceps and place slides in a
Coplin jar fllied 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 DNA-probes according to manufacturers instructions or add 1.0 III of biotin or digoxigenine-Iabeled chromosome specific satellite probes (e.g. Oncor Inc., Vysis, Inc.) plus 0.6 III of IIlg!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 ml50% deionized
formamide/2xSSC/50mM phosphate buffer for 3 h at 70°C. Aliquot
and store at -20°C} (see comment 8).
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 manufactures instructions (e.g. do a prehybridization step at 37°C for 20 min for any kind of library, YACs, BACs,
cosmids or PI-clones).
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 9).