14 Nucleus Extraction from CryofIxed Tissue
181
Fluorescence in situ hybridization (FISH)
1. Add 100 J.lI 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
O:l) to get pH 7.0, then aliquot and store at -20°e].
2. Incubate slides on a warming plate for 12 min at 75°C (see comment 9).
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 add 200 ng of labeled YAC-, BAC- or
cosmid-DNA or 1.5 J.lI of biotin or digoxigenin-Iabeled chromosome
specific satellite probes plus 3 J.lI of 1 J.lg/J.lI COTl-DNA (Gibco) to
20 J.lI of the hybridization-buffer in a 1.5-ml microtube, vortex and
spin down. (Hybridization-buffer: Dissolve 2 g dextran sulfate in
10 ml 50% deionized formamide/2xSSC/50 mM 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 do a
prehybridization step at 37°C for 30 min for the other probes (YACs,
BACs, cosmids).
7. Add 20 J.lI 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 10).
9. Take the slides out of37°C, remove rubber cement with forceps and the
coverslips by letting them swim off in 4x SSC/0.2%Tween (RT, 100-rnl
Coplin jar) (see comment 11).
10. Postwash the slides 3x 5 min in formamide-solution (45°C) followed
by 3x5 min in 2xSSC (37°C) in a 100-ml Coplin jar, with gentle agitation.
181
Fluorescence in situ hybridization (FISH)
1. Add 100 J.lI 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
O:l) to get pH 7.0, then aliquot and store at -20°e].
2. Incubate slides on a warming plate for 12 min at 75°C (see comment 9).
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 add 200 ng of labeled YAC-, BAC- or
cosmid-DNA or 1.5 J.lI of biotin or digoxigenin-Iabeled chromosome
specific satellite probes plus 3 J.lI of 1 J.lg/J.lI COTl-DNA (Gibco) to
20 J.lI of the hybridization-buffer in a 1.5-ml microtube, vortex and
spin down. (Hybridization-buffer: Dissolve 2 g dextran sulfate in
10 ml 50% deionized formamide/2xSSC/50 mM 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 do a
prehybridization step at 37°C for 30 min for the other probes (YACs,
BACs, cosmids).
7. Add 20 J.lI 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 10).
9. Take the slides out of37°C, remove rubber cement with forceps and the
coverslips by letting them swim off in 4x SSC/0.2%Tween (RT, 100-rnl
Coplin jar) (see comment 11).
10. Postwash the slides 3x 5 min in formamide-solution (45°C) followed
by 3x5 min in 2xSSC (37°C) in a 100-ml Coplin jar, with gentle agitation.