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HElKE STEILEN-GIMBEL and STEFFI URBSCHAT
3. Apply the probe to the slide (target area) and cover with a 24x60 mm
coverslip.
4. Seal with rubber cement.
5. Hybridize at 37°C in a moist chamber for 48 h.
If you use commercially available single copy DNA probes (e.g. ONCOR):
First prepare the a-satellite DNA probe.
1. Denature 10 JlI ofthe a-DNA probe dissolved in hybridization mixture
(6 nglJlI) for 10 min at 80°C, place it on ice for 1 min and warm it again
at 37°C.
2. Add the single copy probe to the a-satellite DNA probe, mix well and
spin down for a few seconds.
3. Incubate the probes for 5 min at 37°C.
4. Apply the probe to the slide (target area) and cover with a 24x60 mm
coverslip.
5. Seal with rubber cement.
6. Hybridize at 37°C in a moist chamber for 48 h.
Post hybridization
washes
Signal detection
1. Perform stringency washes in e.g. 50% formamidel2xSSC, pH 7.0 and
in e.g. 2xSSC at 37°C each for 3x5 min (formamide concentration in
the stringency washes depends on the probes).
2. Incubate the slides in 4xSSC/0.05% Tween-20 to adjust to the following signal detection.
1. Apply 120 JlI 4 x SSC/O.5% blocking reagent to each slide to reduce
background.
2. Incubate the slides for 30 min at 37°C in a moist chamber.
3. Apply 120 JlI streptavidin-fluorochrome 2 (e.g. FITC or AMCA) diluted
1:100 in 4xSSC/0.5% blocking reagent to each slide.
4. Incubate for 1 h at 37°C in a moist chamber.
5. Wash briefly 3x5 min with 4xSSC/0.05% Tween-20.
6. Apply 120 JlI of anti-streptavidin-biotin diluted 1:100 in 4xSSC/0.5%
blocking reagent to each slide.