326
THOMAS LIEHR
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 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 5 min at 75°C.
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, precipitate 200 ng of the biotin labeled
probe together with 4 Ilg Cotl-DNA and 20 Ilg herring sperm DNA with
2.5 vol 100% ethanol for 20 min at -80°C or 12-20 h at -20°C (see comment 3).
6. Pellet the DNA by centrifugation at 15,000 rpm for 15 min, discard the
supernatant and dry the DNA pellet at RT or using a speed-vac.
7. Dissolve the pellet from step 6 in 20 III of hybridization-buffer, vortex
and spin down. (Hybridization-buffer: Dissolve 2 g dextran sulfate in
10 ml50% deionized formamide/2x SSC/50 mM phosphate buffer for 3
hat 70°C. Aliquot and store at -20°C).
8. Denature the probe-solution at 75°C for 5 min and do a prehybridization step at 37°C for 30 min.
9. 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).
10. Incubate slides for three nights at 37°C in a humid chamber.
11. Take the slides out of the 37°C humid chamber, remove rubber cement
with forceps and coverslips by letting them swim off in 4xSSC/
0.2%Tween (RT, 100 ml Coplin jar) (see comment 4).
12. Postwash the slides 3 x 5 min in formamide-solution (45°C) followed by
3 x 5 min in 2x SSC (37°C) in a 100-ml Coplin jar, with gentle agitation.
THOMAS LIEHR
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 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 5 min at 75°C.
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, precipitate 200 ng of the biotin labeled
probe together with 4 Ilg Cotl-DNA and 20 Ilg herring sperm DNA with
2.5 vol 100% ethanol for 20 min at -80°C or 12-20 h at -20°C (see comment 3).
6. Pellet the DNA by centrifugation at 15,000 rpm for 15 min, discard the
supernatant and dry the DNA pellet at RT or using a speed-vac.
7. Dissolve the pellet from step 6 in 20 III of hybridization-buffer, vortex
and spin down. (Hybridization-buffer: Dissolve 2 g dextran sulfate in
10 ml50% deionized formamide/2x SSC/50 mM phosphate buffer for 3
hat 70°C. Aliquot and store at -20°C).
8. Denature the probe-solution at 75°C for 5 min and do a prehybridization step at 37°C for 30 min.
9. 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).
10. Incubate slides for three nights at 37°C in a humid chamber.
11. Take the slides out of the 37°C humid chamber, remove rubber cement
with forceps and coverslips by letting them swim off in 4xSSC/
0.2%Tween (RT, 100 ml Coplin jar) (see comment 4).
12. Postwash the slides 3 x 5 min in formamide-solution (45°C) followed by
3 x 5 min in 2x SSC (37°C) in a 100-ml Coplin jar, with gentle agitation.