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HARRY SCHERTHAN
4. Pick up slides with forceps, rinse off coverslips and denaturation solution with a jet of cold deionized water from a wash bottle and air dry.
Note: Dehydration through an alcohol series has proven dispensable.
In situ hybridization
1. Denature hybridization solution for 5 min at 93°C.
2. Place microfuge tube for Ih at 37°C to preanneal any shared repetitive
sequences.
3. Apply an appropriate volume (llli for a round coverslip with 10 mm is
sufficient) to a region of the previously denatured slide with a sufficient
density of metaphase plates.
4. Mount coverslip and seal with rubber cement.
5. Place slides for >96 h in a sparsely moistened chamber or incubator at
37°C.
6. After hybridization peel off rubber cement with forceps.
7. Submerse slides in a Coplin jar with 0.05 x SSC until coverslips float off.
8. Perform post-hybridization washes for 3x5min in 0.05xSSC at 37°C.
Signal detection
Two color fluorescent detection (Fig. 2) is achieved by incubation with
avidin-FITC (Pinkel et al. 1986) and anti digoxigenin rhodamine Fab fragments. In single-color FISH experiments (Fig. 1) either label may be used.
1. Replace the 0.05xSSC (from step 8 above) by 0.5% BSA/BT buffer and
incubate for 5 min at 37°C.
2. Remove slides from Coplin jar, briefly drain excess fluid and place
them in a moist box.
Note: Preparations should never dry up during this and subsequent
steps.
3. Apply 100 III BT buffer containing avidin-FITC (2.5 /lg/ml) and antidigoxigenin rhodamine Fab fragments at 0.5 /lg/ml.
HARRY SCHERTHAN
4. Pick up slides with forceps, rinse off coverslips and denaturation solution with a jet of cold deionized water from a wash bottle and air dry.
Note: Dehydration through an alcohol series has proven dispensable.
In situ hybridization
1. Denature hybridization solution for 5 min at 93°C.
2. Place microfuge tube for Ih at 37°C to preanneal any shared repetitive
sequences.
3. Apply an appropriate volume (llli for a round coverslip with 10 mm is
sufficient) to a region of the previously denatured slide with a sufficient
density of metaphase plates.
4. Mount coverslip and seal with rubber cement.
5. Place slides for >96 h in a sparsely moistened chamber or incubator at
37°C.
6. After hybridization peel off rubber cement with forceps.
7. Submerse slides in a Coplin jar with 0.05 x SSC until coverslips float off.
8. Perform post-hybridization washes for 3x5min in 0.05xSSC at 37°C.
Signal detection
Two color fluorescent detection (Fig. 2) is achieved by incubation with
avidin-FITC (Pinkel et al. 1986) and anti digoxigenin rhodamine Fab fragments. In single-color FISH experiments (Fig. 1) either label may be used.
1. Replace the 0.05xSSC (from step 8 above) by 0.5% BSA/BT buffer and
incubate for 5 min at 37°C.
2. Remove slides from Coplin jar, briefly drain excess fluid and place
them in a moist box.
Note: Preparations should never dry up during this and subsequent
steps.
3. Apply 100 III BT buffer containing avidin-FITC (2.5 /lg/ml) and antidigoxigenin rhodamine Fab fragments at 0.5 /lg/ml.