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HElKE STEILEN-GIMBEL and STEFFI URBSCHAT
14. Wash 3x 5 min with PBS/O.O I%Triton X-I 00.
IS. Fix the slides for 3-5 min in freshly prepared 4% paraformaldehyde
solution (diluted in PBS) on ice.
16. Wash the slides with PBS/O.Ol%Triton X-I00 for 10 min.
Dehydrate the slides in an ethanol series and air dry for the subsequent
FISH procedure.
Multicolor-FISH with a-satellite and/or single copy probes
Probe preparation and hybridization with a-satellite probes:
1. Prepare 30 III of the hybridization mixture containing 65% deionized
formamide, 2 1l120xSSC, 3 III biotinylated DNA probe 1 (20-40 ng/Ill),
and 3 III digoxigenin-Iabeled DNA probe 2 (20-40 ng/Ill).
2. Apply the probe solution onto the immunostained region of the airdried slides, cover with a 24 x 60 mm coverslip, and seal with rubber
cement.
3. Denaturate probe and target DNA simultaneously on a heating plate
for 12 min at 80°e.
4. Incubate the slides at 37°C overnight in a moist chamber.
5. Perform stringency washes in e.g. 65% formamidel2xSSC, pH 7.0 at
37°C for 3x5 min.
6. Incubate the slides in 4xSSC/0.5% blocking reagent (Roche Molecular
Biochemicals) to adjust to the following signal detection.
Probe preparation and hybridization with single copy plus a-satellite
probes:
Pretreatment
of the slides
1. Wash the immunostained slides for 5 min with 2x SSe.
2. Place the slides for 10 min in a pepsin solution (7 mAnson V/100 ml
0.01 M HCI) at 37°e.
3. Wash 2x5 min in 2xSSC at RT.
4. Wash 1x5 min in 1xPBS at RT.