Frequently Asked Questions

Applications

 

Can I perform FISH more than once on the same hybridization zone?

Yes. See protocol section for sequential FISH.

 

What is the smallest size of DNA (contiguous DNA sequence) that can be seen using the Vysis directly labeled probes (CEP or LSI)?

The size depends upon numerous factors; in general, about 30 kilobases is the smallest limit.

 

How do you mix 2 probes together?

Add 7 µL of hybridization buffer, 1 µL of purified water, and 1 uL of EACH probe for a total volume of 10 µL.

 
 
 
 
Filters

 

Which filters are recommended for PathVysion, UroVysion, and AneuVysion?

  • PathVysion: (3 filters) green/orange v.2, DAPI/9-Orange, and green single pass filter. 
  • UroVysion: (4 filters) DAPI, red/green, gold, and aqua. 
  • AneuVysion: (4 filters) DAPI/Orange/Green, green single pass, orange single pass, and aqua single pass. 

For filter information on other products, please contact Vysis Technical Service at 1-800-553-7042 x2.

 

What type of lamp source is optimal for viewing fluorescence?

A 100-Watt mercury lamp source is recommended and should be changed every 200 hours.

 

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.

 

Can a Texas Red filter be used to view Vysis SpectrumOrange probes?

No. The SpectrumOrange probes require the use of a Vysis SpectrumOrange filter. The wavelengths of red and orange are close enough in the spectrum for signals to be seen, but the signal intensity will not be reproducible.

 

Can a gold filter be used to view Vysis SpectrumOrange probes?

No. The SpectrumOrange probes require the use of a Vysis SpectrumOrange filter. The wavelengths of gold and orange are close enough in the spectrum for signals to be seen, but the signal intensity will not be reproducible.

 
 
 
 
General

 

What is the difference between FDA cleared and approved?

FDA approval refers to products that are approved for marketing under the PMA (pre-market approval) process for new devices. FDA clearance refers to devices that are cleared for marketing under a 510 (K) review. Generally the PMA process is more stringent, targeting truly novel products. Devices that are conceptually similar to those already on the market, or that represent improvements over existing products, can elect FDA clearance under a 510(K).

 

Can Vysis offer advice as to how to report a patient result?

No. Vysis is not a clinical laboratory and cannot offer opinions as to how to report a patient result. Vysis can also not offer nomenclature advice.

 

What is the difference between ASR and IVD?

ASR,n — Analyte Specific reagents. ASRs are products for use in home brew testing, which have manufacturer assurances of GMP. ASRs must belabeled in accordance with 21 CFR § 809.10(e). Advertising and promotional materials are regulated by 21 CFR § 809.30(D). The laboratory that develops an in-house test using the ASRs shall inform the ordering person of the test result by appending to the test report this statement according to 21 CFR § 809.30(e): "This test was developed and its performance characteristics determined by (laboratory name). It has not been cleared or approved by the U.S. FDA." ASRs do not have any intended use claims; the laboratory is responsible for establishing intended use and cutoffs. Examples of Vysis ASR product: LSI® bcr/abl, LSI DiGeorge, TotelVysionIVD, In vitro diagnostic use, n — Diagnostic product cleared or approved by the FDA for one or more specific intended uses with established analytical and clinical performance characteristics. Examples of Vysis IVD product: PathVysion, UroVysion, AneuVysion, CEP® 8 SO, CEP 12 SO, CEP X/Y.

 

Why are lot numbers on the vials different than the kit lot numbers?

The lot number that appears on the Certificate of Analysis that is furnished with each probe is the kit lot number. Most kits are comprised of a vial of probe and a vial of hybridization buffer. Other kits contain probe pre-mixed with hybridization buffer. Each of the component parts has a unique part number and lot number separate from those of the kit. When you order the product from our catalog, the number you use is the catalog number, not the individual part numbers that appear on the vials contained within the kit.

 
 
 
 
Probes

 

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.

 
 
 
 
Sample Preparation

 

How should my slides (with metaphase preparations) look prior to doing FISH?

Evaluate your slide preparations under a phase contrast microscope.

  • Good preparation: Chromosomes and nuclei should be dull gray, nonrefractile, and with minimal cytoplasm.
  • Poor preparation: Cells will be "phase bright", appearing white with a halo around the nuclei. Possible cause: Sample on the slides may have dried too quickly during preparation. Solution:
    • Increase the humidity around the slide when "dropping" the sample by placing it on a rack over a 65°C water bath for 20 minutes.
    • Do not bake the slides; baking slides can adversely affect the hybridization results.
    • Dry slides at room temperature overnight. Best results are obtained when the slides are aged overnight at room temperature.
  • Slides prepared and used on the same day are less than optimal, but the following steps can be used to prepare slides in one day: dry the slide first on a 45°C slide warmer for 30 minutes, then dehydrate the slide for one minute each in 70%, 85%, and 100% ethanol solutions prior to the denaturation of the target DNA (step 1 for FISH).

 

After FISH, I have a dim signal and a "haze" covering the slide. How can I correct this?

Usually, the cause is from too high a density of cells dropped onto the slide. The haze is from the cell cytoplasm. Try washing the cell pellet with fresh fixative, dilute the sample and drop onto a new slide. Some types of samples (touch preps, smears of bone marrow or blood,paraffin-embedded samples) may be more prone to a "haze" problem. These types of samples may require additional treatments:

  • Smear: 30 second wash in 70% acetic acid, air dry, 30 second wash in 100% methanol.
  • Paraffin-embedded: increase protein digestion time.

 

Why is the counterstain too faint?

The slides may not have been dried properly before the counterstain was applied. Be sure to age the slides for 24 hours after dropping the cells. Try dehydrating the samples by a series of ethanol washes, then re-counterstain. Also, be sure you have the proper filter for viewing the counterstain. There are two concentrations of DAPI counterstain available from Vysis. DAPI II (125 ng/mL) is for use with CEP® and LSI® probes is an eight-fold dilution of DAPI I (1000 ng/mL) which is for use with WCP® Chromosome Paints. Use of DAPI II with WCP probes may produce too faint a counterstain.

 

Why is the cell morphology of my sample distorted?

Different tissue or sample types may require different denaturation times. If the denaturation time is too long for the sample type, the cells will appear distorted. Try a four minute denaturation time to start and then adjust accordingly.

 

How long can I store my prepared slides and still hybridize them effectively?

If slides are held at -20°C under nitrogen, they should be usable for at least six months.

 

After hybridization, how long can I store my slides and still see fluorescence?

If the slides are stored at -20°C and not exposed too frequently to the high intensity UV light of the mecury lamp, then they will last several months. Signals will fade over time and with light exposure.

 

Can I perform FISH after G banding my metaphase spreads?

Yes, the following reference contains a procedure:

 

JalalSM, Law ME, Christensen ER, Spurbeck JL, Dewald GW. Method forsequential staining of GTLbanded metaphases with fluorescentlabeledchromosome specific paint probes. Am J Med Genet 46:98103, 1993.

 
 
 
 
Reagents

 

Why do the Vysis procedures not call for the addition of antifade to the FISH assay?

The Vysis counterstains, DAPI I Counterstain, DAPI II Counterstain, Propidium Iodide Counterstain, all contain antifade in the solution.

 

The wash solution containing 0.1% NP-40 turns cloudy when I warm it. What is wrong?

Nothing is wrong. The cloudy appearance is normal and will not interfere with the wash.

 

What is the difference between DAPI I and DAPI II?

DAPI I is 1000 ng/mL, about 8X the concentration of DAPI II which is 125 ng/mL. The more concentrated DAPI I works well with WCP® Chromosome Paints and provides a bright visual counterstain for the metaphase chromosomes. The less concentrated DAPI II is for use with the CEP® and LSI® probes which have more compact signals that can be overwhelmed by too much DAPI counterstain of the nuclear DNA.

 

What is the difference between Paraffin Pretreatment Kit I, II, and III?

Paraffin Pretreatment Kit should be used with PathVysion® Her-2 neu tissue samples. It was FDA approved for this kit. It is a more time consuming pretreatment (about an hour longer) than Kit II. Paraffin Pretreatment Kit II is a shorter pretreatment protocol for use on paraffin sections other than for PathVysion Her-2 and prostate tissue. Paraffin Pretreatment Kit III is a more stringent protocol that should be used on prostate tissue or for troubleshooting difficult specimens. This kit contains the enzyme Proteinase K instead of Pepsin.

 

What components make up the hybridization buffers?

  • CEP Hybridization Buffer: 55% formamide, 1XSSC, 10% dextran sulfate
  • LSI/WCP Hybridization Buffer: 50% formamide, 2XSSC, 10% dextran sulfate
 
 
 
 

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