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HORST ZITZELSBERGER, BENJAMIN O'BRIEN, and HEINZ-ULRICH WEIER
Additional material needed for Rx-FISH
- RxFISH Color Chromosome Analysis FISH Kit (Applied Imaging)
- Image recording device (e.g. ISIS, MetaSystems)
- single excitation filters for DAPI, FITC, TRITC and CyS (Zeiss)
Additional material needed for CR-FISH
- Random Priming kit (e.g. BioPrime, Gibco/LTI, Gaithersburg, MD)
- Image recording device (e.g. QUIPS + ScilImage software)
- various filter sets (e.g. triple-and quadruple-color sets, Chroma Technology, Brattleborrow, VT)
Procedure
Slide preparation
Slide preparation should be performed as described in Chapter 3, FISH on
Chromosome Preparations of Peripheral Blood by Thomas Liehr and Uwe
Claussen.
Pretreatment of slides, hybridization and detection of bound probes
Depending on the age and nature of chromosome preparations a pretreatment of slides with pepsin or proteinase K solution might improve the
results. This step is especially recommended, if preparations were not
stored at -20°C or if cytoplasmic residue is visible around the metaphase
spreads under the phase contrast microscope. It is also recommended to
reduce background, which might occur due to proteins around the metaphases. A pepsin treatment is performed before the denaturation step, a
proteinase K treatment after denaturation of preparations.
Pepsin treatment: Slides are incubated in 2x SSC for 1 h at 37°C and are
transferred to prewarmed (37°C) pepsin solution. Incubation may vary
from 1-3 min and digestion of cytoplasm should be checked under phase
contrast microscope (briefly wash in 1x PBS). Slides are washed in 1x PBS
twice, transferred in PBS/MgCI 2 solution for 5 min and fixed in form-
HORST ZITZELSBERGER, BENJAMIN O'BRIEN, and HEINZ-ULRICH WEIER
Additional material needed for Rx-FISH
- RxFISH Color Chromosome Analysis FISH Kit (Applied Imaging)
- Image recording device (e.g. ISIS, MetaSystems)
- single excitation filters for DAPI, FITC, TRITC and CyS (Zeiss)
Additional material needed for CR-FISH
- Random Priming kit (e.g. BioPrime, Gibco/LTI, Gaithersburg, MD)
- Image recording device (e.g. QUIPS + ScilImage software)
- various filter sets (e.g. triple-and quadruple-color sets, Chroma Technology, Brattleborrow, VT)
Procedure
Slide preparation
Slide preparation should be performed as described in Chapter 3, FISH on
Chromosome Preparations of Peripheral Blood by Thomas Liehr and Uwe
Claussen.
Pretreatment of slides, hybridization and detection of bound probes
Depending on the age and nature of chromosome preparations a pretreatment of slides with pepsin or proteinase K solution might improve the
results. This step is especially recommended, if preparations were not
stored at -20°C or if cytoplasmic residue is visible around the metaphase
spreads under the phase contrast microscope. It is also recommended to
reduce background, which might occur due to proteins around the metaphases. A pepsin treatment is performed before the denaturation step, a
proteinase K treatment after denaturation of preparations.
Pepsin treatment: Slides are incubated in 2x SSC for 1 h at 37°C and are
transferred to prewarmed (37°C) pepsin solution. Incubation may vary
from 1-3 min and digestion of cytoplasm should be checked under phase
contrast microscope (briefly wash in 1x PBS). Slides are washed in 1x PBS
twice, transferred in PBS/MgCI 2 solution for 5 min and fixed in form-