31 Multicolor FISH Techniques for the Detection of Inter- and Intrachromosomal Rearrangements
415
aldehyde solution for 5 min at room temperature. Slides are washed in
1x PBS and dehydrated in an ethanol series (70%, 90%, 100%).
Proteinase K treatment: Slides are denatured and dehydrated in an
ethanol series (see above) and transferred to prewarmed (37°C) proteinase
K solution. They are incubated for 7 min and dehydrated again in an ethanol series.
The RxFISH probes are supplied as a ready-to-use hybridization mixture
in a buffer containing formamide. The tube containing RxFISH probes is
warmed in a 37°C water bath for 5 min. An aliquot of 10 J-ll sufficient for a
single hybridization is placed in a fresh tube and denatured by incubating
at 65°C for 10 min. The remaining RxFISH probes are kept at -20°e. The
metaphase preparations are denatured at 70-75°C in 70% FA/2xSSC, pH
7.0 for 1.5-2 min. This step may require modification according to the type
and age of samples. Older slides often require longer denaturation times.
The denatured slides are immediately placed in ice-cold 70% ethanol for
2 min. Then, dehydrate the slides in the following ethanol series for 2 min
each at room temperature: 70%, 2x 90%, 100% and air dry. The 10 J-ll
denatured probe mixture is applied to the slide, which is covered by a
22 x 22 mm coverslip and sealed with rubber cement. The hybridization
proceeds for 12-72 h in a moist chamber.
After hybridization, the slide is washed in 2x SSC, twice in 50% formamide/O.5 x SSC, twice in 2x SSC and in 4 x T solution at 45°C for 5 min each.
The last washing step is performed for 10 min. For signal amplification of
the FITC-labeled probe, the antibody solutions F1 and F2 (provided with
the Kit) are centrifuged at 12,000 rpm for 10 min at room temperature and
200 J-li of F1 is pipetted onto the slide, covered with a ParafIlm coverslip
and incubated at 37°C for 30 min in a moist chamber. After six washes in
4xT solution at 42°C this step is repeated with F2. After the last wash in
4x T solution, the slide is counterstained in DAPI counterstain solution
for at least 2 min and mounted with 25 J-li mounting media Citifluor AFI.
The PI DNAs are extracted from overnight cultures following an alkaline
lysis protocol (Weier et aI, 1995b; Chapter 18, Quantitative DNA Fiber
Mapping by Heinz-Ulrich Weier). The YAC DNAs are isolated from
48-h liquid cultures following a standard protocol using yeast lytic enzyme
(ICN, Aurora, OH) (Weier et al 1995b; Chapter 18, Quantitative DNA
Fiber Mapping by Heinz-Ulrich Weier). The DNAs are then precipitated
with isopropanol, washed once in 70% ice cold ethanol and resuspended
in 1x TE buffer. To further improve purity, the DNA is extracted with
phenollcWoroform mixture, again precipitated in isopropanol, resusHybridization
for RX-FISH
Hybridization
for CR-FISH
415
aldehyde solution for 5 min at room temperature. Slides are washed in
1x PBS and dehydrated in an ethanol series (70%, 90%, 100%).
Proteinase K treatment: Slides are denatured and dehydrated in an
ethanol series (see above) and transferred to prewarmed (37°C) proteinase
K solution. They are incubated for 7 min and dehydrated again in an ethanol series.
The RxFISH probes are supplied as a ready-to-use hybridization mixture
in a buffer containing formamide. The tube containing RxFISH probes is
warmed in a 37°C water bath for 5 min. An aliquot of 10 J-ll sufficient for a
single hybridization is placed in a fresh tube and denatured by incubating
at 65°C for 10 min. The remaining RxFISH probes are kept at -20°e. The
metaphase preparations are denatured at 70-75°C in 70% FA/2xSSC, pH
7.0 for 1.5-2 min. This step may require modification according to the type
and age of samples. Older slides often require longer denaturation times.
The denatured slides are immediately placed in ice-cold 70% ethanol for
2 min. Then, dehydrate the slides in the following ethanol series for 2 min
each at room temperature: 70%, 2x 90%, 100% and air dry. The 10 J-ll
denatured probe mixture is applied to the slide, which is covered by a
22 x 22 mm coverslip and sealed with rubber cement. The hybridization
proceeds for 12-72 h in a moist chamber.
After hybridization, the slide is washed in 2x SSC, twice in 50% formamide/O.5 x SSC, twice in 2x SSC and in 4 x T solution at 45°C for 5 min each.
The last washing step is performed for 10 min. For signal amplification of
the FITC-labeled probe, the antibody solutions F1 and F2 (provided with
the Kit) are centrifuged at 12,000 rpm for 10 min at room temperature and
200 J-li of F1 is pipetted onto the slide, covered with a ParafIlm coverslip
and incubated at 37°C for 30 min in a moist chamber. After six washes in
4xT solution at 42°C this step is repeated with F2. After the last wash in
4x T solution, the slide is counterstained in DAPI counterstain solution
for at least 2 min and mounted with 25 J-li mounting media Citifluor AFI.
The PI DNAs are extracted from overnight cultures following an alkaline
lysis protocol (Weier et aI, 1995b; Chapter 18, Quantitative DNA Fiber
Mapping by Heinz-Ulrich Weier). The YAC DNAs are isolated from
48-h liquid cultures following a standard protocol using yeast lytic enzyme
(ICN, Aurora, OH) (Weier et al 1995b; Chapter 18, Quantitative DNA
Fiber Mapping by Heinz-Ulrich Weier). The DNAs are then precipitated
with isopropanol, washed once in 70% ice cold ethanol and resuspended
in 1x TE buffer. To further improve purity, the DNA is extracted with
phenollcWoroform mixture, again precipitated in isopropanol, resusHybridization
for RX-FISH
Hybridization
for CR-FISH