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JINGLY FUNG et at.
a negative impact on human development, the preimplantation and prenatal genetic diagnosis tests should score as many of the full complement
of chromosomes as possible. This chapter describes a procedure for the
enumeration of multiple chromosomes in interphase cells using Spectral
Imaging (SIm)(Fung 1998). In interphase analysis, the use of whole chromosome painting probes is inappropriate because the chromatin domains
of chromosomes overlap each other in the less condensed nucleus. Consequently, chromosome- or locus-specific probes for interphase analysis
must be developed. Moreover, careful cell preparation and pretreatment
are essential to ensure high hybridization efficiency and minimal signal
overlap. This chapter describes in detail the techniques for the preparation
of metaphase and interphase cell slides, the preparation of locus-specific
probes, hybridization and detection, imaging acquisition and analysis
using the Spectral Imaging system.
Outline
- Grow, swell and fix cells
- Prepare cell spreads on microscope slides
- Select probe set
- Pretreat slides
- Denature and hybridize probe set to cellular targets
- Remove unbound probe
- Detect bound probes with antibodies
- Acquire images with a Spectral Imaging system
- Analyze images with a library of pre-recorded spectra
- Display results in comprehensive and informative format
Materials
Equipment - CCD camera (Hamamatzu)
- CDS-5 Cytogenetic Drying Chamber (Thermotron Industries)
- Centrifuge (MP4R, IEC)
JINGLY FUNG et at.
a negative impact on human development, the preimplantation and prenatal genetic diagnosis tests should score as many of the full complement
of chromosomes as possible. This chapter describes a procedure for the
enumeration of multiple chromosomes in interphase cells using Spectral
Imaging (SIm)(Fung 1998). In interphase analysis, the use of whole chromosome painting probes is inappropriate because the chromatin domains
of chromosomes overlap each other in the less condensed nucleus. Consequently, chromosome- or locus-specific probes for interphase analysis
must be developed. Moreover, careful cell preparation and pretreatment
are essential to ensure high hybridization efficiency and minimal signal
overlap. This chapter describes in detail the techniques for the preparation
of metaphase and interphase cell slides, the preparation of locus-specific
probes, hybridization and detection, imaging acquisition and analysis
using the Spectral Imaging system.
Outline
- Grow, swell and fix cells
- Prepare cell spreads on microscope slides
- Select probe set
- Pretreat slides
- Denature and hybridize probe set to cellular targets
- Remove unbound probe
- Detect bound probes with antibodies
- Acquire images with a Spectral Imaging system
- Analyze images with a library of pre-recorded spectra
- Display results in comprehensive and informative format
Materials
Equipment - CCD camera (Hamamatzu)
- CDS-5 Cytogenetic Drying Chamber (Thermotron Industries)
- Centrifuge (MP4R, IEC)