How stable are the probes? Can I freeze-thaw them? Can I store them diluted?
When stored properly, the probes are stable. They can be repeatedly frozen and thawed. There is less risk of degradation of the probes if they are stored undiluted.
Do I need to amplify the signal?
No, signal amplification is not necessary. The Vysis directly labeled probes have bright signal without any additional detection steps.
Can I decrease the amount of probe per assay?
No, the signal strength will be too weak.
What is the difference between directly labeled and indirectly labeled probes?
With directly labeled probes, the fluorophore is covalently linked to the DNA probe. With indirectly labeled probes, a hapten, such as biotin or digoxigenin, is incorporated into the probe. A series of posthybridization steps ("sandwiching") allows a fluorophore to be bound to the happen.
When do I use WCP vs CEP probes?
CEP Probes are used on both interphase cells and metaphase spreads and can be used to determine ploidy (e.g. trisomy 18). WCP Whole Chromosome Painting probes are used on metaphase spreads, only. Use on interphase cells is not recommended by the scientific community, due to difficulties in interpretation of results.
Do the WCP probes cross-react to other human chromosomes or to rodent chromosomes?
Some cross-reactivity with other human chromosomes may occur with the WCP probes. The probe mixture contains human Cot-1®* DNA to reduce binding to repeated sequences on the target and non-target DNA. You may add more human Cot-1 DNA. Some of the WCP probes are prepared form DNA libraries of flowsorted human chromosomes obtained from human/hamster somatic cell hybrids. Use GIBCO/BRL Hamster Cot1 DNA or Mouse Cot1 DNA as a blocking reagent if you are analyzing somatic cell hybrids for their human chromosome content.
If mixing LSI and CEP probes, which hybridization buffer is used?
Use LSI hybridization buffer. The CEP buffer is the more stringent, the LSI buffer the less stringent. The LSI requires the less stringent buffer or it may not hybridize to its target. Not all configurations of combining LSI and CEP have been tested by Vysis. It is possible that for some probe mixes, the CEP probe may experience cross-hybridization with other chromosomes due to the lower stringency conditions.
How do you mix 2 probes together?
Add 7 µL of hybridization buffer, 1 µL of purified water, and 1 µL of EACH probe for a total volume of 10 µL.
Why are my fluorescent signals faint?
In addition to using the appropriate filter, the microscope excitation source and age of the lamp will affect how strong the fluorophore signal appears. We recommend a 100 watt mercury lamp with less than 200 hours used. Some probe signals are brighter or have different shapes when compared to other probe signals. For example, an LSI signal is more compact than a CEP signal and a viewer may interpret this as a less bright signal. In fact, the signal for the LSI is just smaller. Different WCP Chromosome Paints have varying staining intensities and a viewer may describe a particular WCP Chromosome Paint as not as bright as the previous one they were using. Compared to some amplified signals of indirectly labeled probes, the signal from directly labeled probes may be described by some viewers as faint. Vysis probe signals may appear smaller, but the signal is as bright. Also, due the lack of background fluorescence, the signal is easier to see and distinguish as a true signal.