Question Master

If you are experiencing issues with your FISH probes or protocol the FAQ section may feature the solution to your problem. If you do not find a solution within these pages please do not hesitate to contact us directly via our ask the lab option.

• What are the storage and shipping temperatures of Cytocell's products?
  All of our products ship in ice packs (4°C) and can stand at room temperature for up to 12hrs. It is important that the products are NOT thawed, then refrozen below 0°C and then thawed again during shipping, if the items thaw during shipping its advisable to leave them in the thawed state or at 4°C until delivery to the laboratory. On receipt of the products at the laboratory they should be stored at -20°C. Multiprobe products should not be frozen.
• What are the correct filter characteristics which would allow simultaneous visualisation of green, red and blue?
  Currently, there are two different triple filters in common use in laboratories, one which is specific for TRITC/FITC/DAPI and one which allows visualisation of Texas Red/FITC/DAPI. The first one is easily distinguished by the green light that comes from the objective on the microscope, the other gives a white/pale yellow colour. The only one that allows simultaneous visualisation of Cytocell's Green, red and blue dyes is the Texas red/FITC/DAPI filter.
• Can Spectrum Orange and Spectrum Red filters be used to see Cytocell's Red Probes?
  Both types of filter can be used, but using the Spectrum Red will give brighter signals for Texas Red probes and using the Spectrum Orange will give brighter signals for our Orange fluorophore.
• How important is the denaturation temperature?
  It is vital. The probes which are specific for repeat elements of the human genome are less susceptible to over-denaturation than those specific for single sequences of DNA. They can tolerate much more loss in activity as there are more repeat units to which the probes can hybridise. However, one must also consider the sample. This is made of DNA too, and will lose significant levels of morphology if overdenatured. It is always good practice to take good care when denaturing any FISH product to ensure good signals and good sample morphology.
• We are having problems visualising the probes in square 2 (12 centromere) in the CLL panels.
  We use a Vysis Hybrite heating block.
  
  Using Hybrite: Moisten the two strips of paper towel with deionised water (should not be dripping wet) and place the towels in the troughs along the heating surface. Make sure the towels are not protruding above the heating surface and come directly into contact with any slides. Cover spare spaces with empty slides. Prewarm the Hybrite to 37°C with the lid closed. Transfer slides inside and run Cytocell recorded programme: denaturation/hybridisation.
• How big are Cytocell's probes?
  All our probe products are optimised to make them suitable for FISH. We generally cut all our probes up to fragments of between 200 and 2000bp depending on the product. But the size of the signal depends on the length of the DNA sequence recognised by the probe when all fragments recognise their target.
• How long do the high pressure mercury microscope bulbs last?
  At Cytocell we never use a bulb for longer than 200 hours. The older the bulb, the dimmer the fluorescence signals become, and the greater the chance of the bulb exploding. This is dangerous as mercury vapour is highly toxic.
• We had some "AQUARIUS probes" delivered to our lab a long time ago and someone just placed box in our freezer, they have now expired and I am not sure how to dispose of them. The insert that was with it makes it sound like this product is very hazardous. Could you help me figure out how to dispose of it safely?
  We would suggest that you dispose of your unwanted probes by throwing them in the general lab waste; treating the probes as you would any other low-grade hazardous waste. Please note that the probes should not be flushed down a drain.
• I have experienced weak signals when using an SRY deletion probe. I think it may be due to a washing step error, just to clarify we use tween 20 in 2XSSC for 15 sec at 37°C? Please let me know in case I need to modify a step?
  The correct washing procedure is 0.4xSSC at 72'C for 2 minutes, then 2xSSC/0.05% Tween for 30s at room temperature. If you are using 2xSSC at 37 instead of the first hot wash, she should get bright signal and horrible background, rather than weak signal. The problem is more likely to be in the denaturation or with the microscope than the washes: it's hard to go wrong with the washes and lose the signal (unless you go weaker than 0.4xSSC).
• How should tissue samples be stored prior to performing PETS FISH?
  Only tissues preserved in 10% Neutral Buffered Formalin (NBF) and subsequently paraffin embedded or previously paraffin embedded sections are suitable for use.
  
  NBF is most commonly used to preserve tissues and should be used for no longer than 3 months and then discarded. Extended formalin fixation time can cause major difficulties in PETS FISH such as reduced probe penetration, high levels of tissue auto-fluorescence, cell overlap or truncated nuclei and low hybridisation efficiency. Specimens should be fixed for between 18-24h in NBF, tissues fixed for longer will require Pepsin digestion times to be increased.
• How thick should tissues specimens be to perform PETS FISH?
  Tissues specimens should be 4-6μm thick. If sections are too thick, this can introduce difficulties in interpreting signals in different focal planes, distinguishing individual nuclei and poor hybridisation efficiency.
• Is pepsin treatment only required on late gestational age prenatal specimens?
  Pepsin treatment always improves FISH results, regardless of cell type used - lymphocytes, bone marrow, amniocytes, sputum, etc. Cytocell does not usually recommend pepsin treatment for every product because it is time consuming and requires additional costly reagents and for 'regular' cytogenetics preparations the benefits do not warrant the extra work.
  
  We especially suggest that pepsin treatment be used for late gestational age samples because we have noticed that the improvement is marked and certainly worth the additional effort. This does not mean that pre-treatment is essential or only required for late gestational samples, however it can be beneficial as some people find it difficult to analyse FISH results without pre-treatment.
  
  If you already routinely perform pepsin digestion for all samples then maintain this practice as it will produce the best results.
• What is early and late gestation?
  Amniocentesis is routinely performed between 14 and 20 weeks gestation so late gestational age samples are those that are closer to the 20th week. The later in the pregnancy, the more cell debris there is in the amniotic fluid and therefore the greater the advantage you will see from pre-treatment protocols.
• Are the prenatal probes designed for use on paraffin embedded tissue, abortive tissue or placenta? If so which protocol should be used?
  Our Prenatal range is developed for use with uncultured amniocytes so product performance with other cell types cannot be guaranteed. However if the intention is to use prenatal probes on such tissue sections, a solid tumour pre-treatment protocol for paraffin embedded tissue sections could be used, including removal of paraffin wax (de-paraffination), rehydration, acid treatment, pre-treatment and protein digestion.
  
  For cells fixed in Carnoy's fixative, the Prenatal pre-treatment protocol should be followed.
  
  In all cases where the prenatal probes are used for samples not recommended by Cytocell, the recommended protocol should be followed first to establish expected probe brightness and hybridisation efficiency before deviations from the protocol are considered. Cytocell can only guarantee results on recommended tissue types, however.