16 Comparative Genomic Hybridization
209
2. Dry the pellet and resuspend by vortexing vigorously in 7 III MM 1 and
3 III distilled water. Recentrifuge 15 s in the microfuge.
1. Inspect the slide to make certain there are enough (more than five)
metaphases to evaluate. There should be little or no cytoplasm around
the nuclei or metaphases. Outline these regions using a diamond pen.
2. Incubate no more than three slides at a time for 3-8 min in denaturing
solution prewarmed to 75°C. Slides that have been aged for longer periods at room temperature or stored for longer periods, require longer
denaturation times.
3. Dehydrate slides with successive 2 min incubations in 70%, 80%, 90%,
and 100% ethanol, air dry, and preincubate for 1-2 min on a slide warmer set to 37°C.
Prepare metaphase
slides immediately
before
hybridization
1. Denature the hybridization mix in an open 80°C water bath for 6 min. Hybridization
2. Apply 10 III of the hybridization mix immediately to the marked area of
the metaphase slide while on the slide warmer. Remove air bubbles by
touching them with the corner of a 22 mm
2
coverslip.
3. Cover each slide with two coverslips. Use a wide-bore pipette or small
syringe to seal the edges of the coverslips with rubber cement. Allow
5 min to set.
4. Incubate at 37°C for 1-2 days in a covered humid hybridization chamber.
5. Remove rubber cement from hybridized slides and remove cover slips
by placing in 2x SSC (or allow to fall off in first wash).
1. Wash at 45°C: Three times; each 10-15 min in 50% formamide/2x SSC Washes
Two times; each 10 min in 2x SSC
1. Incubate hybridized metaphase spread in blocking buffer for 5 min at Signal amplification
room temperature.
2. Incubate in 1 Ilg/ml rhodamine-conjugated anti-DIG-antibody
(Roche) in blocking buffer for 45 min at room temperature if the control is directly labeled. If the control is labeled with biotin, then simultaneously incubate with both the anti-DIG and avidin-FITC (Boehringer/Mannheim).
209
2. Dry the pellet and resuspend by vortexing vigorously in 7 III MM 1 and
3 III distilled water. Recentrifuge 15 s in the microfuge.
1. Inspect the slide to make certain there are enough (more than five)
metaphases to evaluate. There should be little or no cytoplasm around
the nuclei or metaphases. Outline these regions using a diamond pen.
2. Incubate no more than three slides at a time for 3-8 min in denaturing
solution prewarmed to 75°C. Slides that have been aged for longer periods at room temperature or stored for longer periods, require longer
denaturation times.
3. Dehydrate slides with successive 2 min incubations in 70%, 80%, 90%,
and 100% ethanol, air dry, and preincubate for 1-2 min on a slide warmer set to 37°C.
Prepare metaphase
slides immediately
before
hybridization
1. Denature the hybridization mix in an open 80°C water bath for 6 min. Hybridization
2. Apply 10 III of the hybridization mix immediately to the marked area of
the metaphase slide while on the slide warmer. Remove air bubbles by
touching them with the corner of a 22 mm
2
coverslip.
3. Cover each slide with two coverslips. Use a wide-bore pipette or small
syringe to seal the edges of the coverslips with rubber cement. Allow
5 min to set.
4. Incubate at 37°C for 1-2 days in a covered humid hybridization chamber.
5. Remove rubber cement from hybridized slides and remove cover slips
by placing in 2x SSC (or allow to fall off in first wash).
1. Wash at 45°C: Three times; each 10-15 min in 50% formamide/2x SSC Washes
Two times; each 10 min in 2x SSC
1. Incubate hybridized metaphase spread in blocking buffer for 5 min at Signal amplification
room temperature.
2. Incubate in 1 Ilg/ml rhodamine-conjugated anti-DIG-antibody
(Roche) in blocking buffer for 45 min at room temperature if the control is directly labeled. If the control is labeled with biotin, then simultaneously incubate with both the anti-DIG and avidin-FITC (Boehringer/Mannheim).