Shipping temperatures are dependent on both the product and the destination.

1. CytoCell FISH probes shipped within the EU are shipped at ambient temperature.
2. CytoCell FISH probes shipped outside the EU are shipped at 4°C with an ice pack.
3. Tissue Pretreatment kits are shipped at 4°C.

1. Storage temperatures are dependent on the product.
2. On receipt of the products at the laboratory, CytoCell probes should be stored between -15°C to -25°C.
3. Tissue Pretreatment kits are stored at 2-8°C.

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.

The number provided on the kit is the kit lot number. The individual component(s) in the kit will also have their own lot numbers specific for the manufacturing of those products.

Figure 1. Kit Lot Number

Figure 2. Reagent Lot Number

Please inform CytoCell technical support if you suspect that your probe is showing signs of light damage. Our manufacturing team maintains a probe archive which can be tested internally. The common characteristics of light damaged probes are nonspecific probe binding and high autofluorescence.

To mitigate the effects of light damage, we recommend the following:

• Reduce the level of probe exposure. Cover probe, place in -20°C freezer when not in use.
• Aliquot probe on arrival into single use ‘shots’.

Example 1 - Light damaged probe

Example 2 - Light damaged probe

The expiry date on the Certificates of Analysis (CoA) and DAPI is displayed in YYYY-MM-DD format on both the component tube and the outer kit label.

Figure 1. DAPI tube label

The expiry date of all other catalog products is displayed in YYYY-MM format. Please note this extends to the last day of the month, regardless of date of manufacture on the CoA.

Figure 2. Probe tube label

There should not be a mismatch between the expiry date on the probe label and the label on the outer kit. If you have checked the information above and are still concerned that you may have a mismatch in expiry dates, please contact our support team.

CytoCell probes that begin with the catalog classification LPH correspond to hematology FISH probes and those that begin with LPS correspond to solid tumor/hematopathology FISH probes.

The off-label use of probes is not recommended by CytoCell, the recommended protocol should be followed first to establish expected probe brightness and hybridization efficiency before deviations from the protocol are considered. CytoCell can only guarantee results on the intended sample type.

Hematology probes have been optimized for use on 3:1 methanol/acetic acid fixed peripheral blood or 3:1 methanol/acetic acid fixed bone marrow.

Solid tumor probes have been validated for use on formalin-fixed paraffin-embedded (FFPE) tissue.

Hemato-pathology probes are validated for dual use on 3:1 methanol/acetic acid fixed bone marrow, 3:1 methanol/acetic acid fixed peripheral blood and FFPE tissue.

Pepsin treatment always improves FISH results, regardless of cell type used -peripheral blood, 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 a marked improvement 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.

A solution of 3:1 Methanol/Acetic Acid (Carnoy’s) should be made freshly prepared, stored at -20°C and discarded after a day.

Try pre-fixing your bone marrow or peripheral blood samples by slowly adding ice-cold fix with agitation, immediately after the hypotonic treatment, to improve your cell preparations.

The optimal conditions for spreading are: 25°C and 50% relative humidity (RH).

Recommendations:

• Ideally use a Thermotron^®, as it provides optimal conditions for sample spreading.
• Alternatively, suspend slide over a sink of hot water, or a 37°C water bath, then spot sample.
• Critical for all samples including peripheral blood.

In general, baking slides using a hotplate and/or ageing will produce weaker, more variable results. This tends to be part of older FISH protocols.

CytoCell probes (aside from Sub-telomeric and enumeration probes) are supplied ready to use with probe and hybridization solution pre-mixed. This mix has been developed and optimized for the intended purpose using 10μl of probe and a 24mm x 24mm coverslip. For this reason, we do not recommend dilution or reduction in volume. Both can produce weaker, more variable probe signals.

Please see the example images below for guidance:

Example 1: IGH Plus Breakapart (LPH 070) using 10ul of probe.

Example 2: IGH Plus Breakapart (LPH 070) using 1ul of probe.

Example 3: IGH Plus Breakapart (LPH 070) using 5ul probe + 5ul Cytocell Hybridization A (HA500L)

Recommendations:

• Use a glass coverslip. Avoid using alternatives such as parafilm, clingfilm, etc. These will not create a sufficient seal to allow for probe spreading.
• Ensure coverslips are sealed to prevent evaporation which can lead to weak, diffuse green signals.
• Seal correctly with rubber glue solution e.g. Weldite/Fixogum.

Example 1: P53 (LPH 017) sealed with a glass coverslip and Weldite glue

Example 2: P53 (LPH 017) sealed with Parafilm

Example 3: P53 (LPH 017) sealed with Cling Film

Example 4: P53 (LPH 017) not sealed with glue.

When using the enzyme solution to digest your FFPE slide, liberally apply the enzyme ensuring the section is covered, as evaporation may cause the enzyme to recede from the edges and cause inconsistent digestion. When not in use, ensure that the enzyme solution is stored correctly at 2-8°C to preserve the enzyme activity.

Ensure pipette tips are long enough so that when the tip reaches the bottom of the vial, the pipette itself doesn’t come into contact. Pipettes entering a probe vial may carry over probe reagent from one vial to the next resulting in cross contamination.

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 hybridize. 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.

Figure 1: Example of overdenaturation using Subtelomere 17p and 17q probes with a denaturation of 95°C for 2 mins on cultured lymphocytes.

Figure 2: Example of undenaturation using the MDM2 probe, with a denaturation of 60°C for 2 mins on a skin biopsy sample.

This is likely to be caused by low humidity during the hybridization step. The FITC (green) fluorophore is more sensitive to these changes and can produce loss of or drop out of control probe signals. The optimal humidity levels for efficient probe hybridization are 65-70%; however, we would recommend 95-100% humidity to minimize the risk of weak control signals.

Post-hybridization washing after FISH hybridization is necessary to aid the removal of non-specific interactions between the probe and undesirable regions of the genome, thus allowing greater probe specificity.

The buffers used in post-hybridization washing are SSC based and therefore provide positively charged sodium ions in solution. These ions counteract the repulsive negative force between the DNA backbones of both the probe and target. Too much SSC in the buffer will produce a poor washing effect of low stringency and too little SSC (or just water) will tend to wash all probe away from the sample due to high stringency. Temperature and pH also influence the washing effect; increasing the temperature increases the stringency and the pH determines the availability of the positive ions.

The inclusion of TWEEN 20 detergent decreases background staining and enhances the spreading of the reagents in the wash buffers.

Post hybridization washes at CytoCell are optimized for the FISH procedure and have been formulated using the principles outlined above.

• 0.4xSSC 2 mins washes typically at 72+/-1C, 2xSSC/0.05% Tween for 30s at room temperature is optimal for most probes.
• 0.25xSSC 2 mins washes typically at 72+/-1C, 2xSSC/0.05% Tween for 30s at room temperature for most enumeration probes.

An additional tip would be to periodically wash solution jars and ensure adequate detergent removal can help reduce background issues. Also, the use of filtered pipette tips can reduce intermittent background issues from debris being expelled onto FISH slides.

Figure 1: Example of high stringency >73°C, <0.4xSSC, pH differs from 7.

Figure 2: Example of low stringency <71°C, >0.4xSSC, pH differs from 7. 

This is a common problem particularly with older or well-used hybridizers, where temperature discrepancies are noticeable on the bottom and top of the heating plate. It is not uncommon to have a position that reads at 60°C denaturation despite the program settings stating 75°C. We recommend regular calibration of hybridizers.

Once the DAPI counterstain and coverslip have been applied to the slide, it is recommended that users seal the coverslip with varnish. This prevents excessive drying out and maintains the probe signal strength. For short term storage (0-2 weeks), the slides should be covered and stored at room temperature. For long term storage, the slides should be stored 2-8°C and covered.

Currently, there are two different triple filters commonly used in laboratories, one which is specific for TRITC/FITC/DAPI and one which allows visualization 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 filter that allows simultaneous visualization of CytoCell's green, red and blue dyes is a Texas Red/FITC/DAPI filter.

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.

Depending on the manufacturer’s recommendations, fluorescent filters typically need to be replaced every 2-4 years. Over time and use, the filter will begin to degrade. The damaged emission/excitation filters will show a mottled appearance, which begins on the edges and will eventually completely obscure the filter. The effects may not be noticeable on a day to day basis, but will manifest as higher than normal background and weaker signals.

Please contact CytoCell technical support for further advice and guidance if you suspect filter problems.

Depending on the type of bulb used, the shelf life can vary from 200-3000 hours. Please verify with your supplier. The older the bulb, the dimmer the fluorescence signals become, and the greater the chance of the bulb exploding. This is dangerous as mercury vapor is highly toxic.

Tissues specimens should be 4μm-6μm thick for optimal results. If sections are too thick, this can introduce difficulties in interpreting signals in different focal planes, distinguishing individual nuclei and poor hybridization efficiency. If sections are too thin (<4μm), this can lead to signal truncation.

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 hybridization efficiency. Specimens should be fixed for between 18-24h in NBF, tissues fixed for longer will require pepsin digestion times to be increased.