410
HORST ZITZELSBERGER, BENJAMIN O'BRIEN, and HEINZ-ULRICH WEIER
Outline
- Optional: Irradiate peripheral lymphocytes in vitro or use peripheral
blood of an exposed subject or cultivated tumor cells
- Treat cells with hypotonic solution and fIx
- Prepare metaphase spreads
- Choose DNA probes (Rx or CR probes)
- Pretreat slides (optional for older preparations)
- Denature and hybridize probes with metaphase preparations
- Remove unbound probes
- Score slide and select rearranged metaphases
- Acquire images with an image analysis system
- Describe inversions by comparing FISH patterns and DAPI banding
Note: Caution: Most subprotocols should be seen as suggestions. Typically, previously established procedures should be integrated (image
analysis system, labeling systems, etc.).
Materials
Equipment - Centrifuge (Heraeus, Biofuge fresco)
- CO 2 incubator
- Dry bath (heat block) (Liebisch)
- Fluorescence microscope equipped with 40 x and 63 x oil immersion,
lenses and motorized fIlter changer (Zeiss, Axioplan 2)
- Fluorimeter (Hoefer, DyNA Quant 200)
- Incubator oven (Memmert)
- Phase contrast microscope (Zeiss, Axioskop)
- Thermal cycler for in vitro DNA amplifIcation (model 4800, Perkin
Elmer)
- Water bath (GFL)