12 Nucleus Extraction from Formalin Fixed/paraffin Embedded Tissue
167
incubate at 37°C for about 20 min; then add 50 JlI of the pepsin stock
solution 10% (w/v) (Serva 31855) and leave the Coplin jar at 37°C;
make fresh as required).
7. Replace fluid with 100 mIl xPBS/MgCh, incubate at RT for 5 min with
gentle agitation. MgCh will block the enzymatic activity of pepsin
(1 x PBS/ MgCh =5% (v/v) 1M MgCh in 1xPBS).
8. Postfix nuclei on the slide surfaces by replacing 1x PBS/MgCh with 100
ml of formalin-buffer (3 ml acid free formaldehyde [37%; Roth 4979.1]
in 100 ml lxPBS) for 10 min (RT, with gentle agitation)..
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 5).
Fluorescence in situ hybridization (FISH)
1. Add 100 JlI denaturation-buffer to the slides and cover with (24 x 50
mm) coverslips. (Denaturation-buffer: 70% (v/v) deionized formamide, 10% (v/v) ftltered double distilled water, 10% (v/v) 20xSSC,
10% (v/v) phosphate buffer; make fresh as required; phosphate buffer:
prepare 0.5 M Na2HP04 and 0.5 M NaH 2 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 12 min at 75°C (see comment 6).
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 JlI of biotin or digoxigenin-Iabeled chromosomespecific satellite probes plus 3 JlI of 1 Jlg/JlI COTl-DNA (Gibco) to 20 JlI
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/2 x SSC/50 mM phosphate buffer for 3 h
at 70°C. Aliquot and store at -20°C.)
167
incubate at 37°C for about 20 min; then add 50 JlI of the pepsin stock
solution 10% (w/v) (Serva 31855) and leave the Coplin jar at 37°C;
make fresh as required).
7. Replace fluid with 100 mIl xPBS/MgCh, incubate at RT for 5 min with
gentle agitation. MgCh will block the enzymatic activity of pepsin
(1 x PBS/ MgCh =5% (v/v) 1M MgCh in 1xPBS).
8. Postfix nuclei on the slide surfaces by replacing 1x PBS/MgCh with 100
ml of formalin-buffer (3 ml acid free formaldehyde [37%; Roth 4979.1]
in 100 ml lxPBS) for 10 min (RT, with gentle agitation)..
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 5).
Fluorescence in situ hybridization (FISH)
1. Add 100 JlI denaturation-buffer to the slides and cover with (24 x 50
mm) coverslips. (Denaturation-buffer: 70% (v/v) deionized formamide, 10% (v/v) ftltered double distilled water, 10% (v/v) 20xSSC,
10% (v/v) phosphate buffer; make fresh as required; phosphate buffer:
prepare 0.5 M Na2HP04 and 0.5 M NaH 2 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 12 min at 75°C (see comment 6).
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 JlI of biotin or digoxigenin-Iabeled chromosomespecific satellite probes plus 3 JlI of 1 Jlg/JlI COTl-DNA (Gibco) to 20 JlI
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/2 x SSC/50 mM phosphate buffer for 3 h
at 70°C. Aliquot and store at -20°C.)