Fish 'n' Tips

When not in use, ensure that the enzyme solution is stored correctly at 2-8°C to preserve the enzyme activity.

Keep light exposure of probes to a minimum as this can cause photodegradation.

Use a working reagent box that you take from the freezer instead of all of the probes to minimise light exposure/freeze thaws. Place the box back in the freezer as soon as you remove the probes you need for the day.

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.

If your cells appear intact with a three-dimensional look to them, and there is visible cytoplasm using a phase contrast microscope, you could try re-fixing your cells in 1:1 methanol:acetic acid.

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.

Try not to push down too hard on the coverslip when applying the FISH probe, as it can squeeze out of the sides and create a patchy hybridisation.

To avoid patchy hybridisations, make sure to press out visible bubbles when applying the coverslip on the probe mix.

After pre-treatment, consider using DAPI to assess for over/under digestion of FFPE sections; this can be washed off before applying the FISH probe.

Use calibrated temperature-measuring devices to regularly QC hybridisation units; there may be significant slot to slot variability; this can get worse over time leading to poor results.

Test ramp up and ramp down temperature times periodically as part of equipment QC on automated hybridisation units, to ensure they are within manufacturer specifications.

Test ramp up and ramp down temperature times periodically as part of equipment QC on automated hybridisation units, to ensure they are within manufacturer specifications.

Maintaining humidity strips on automated hybridisation units according to manufacturer recommendations can help ensure optimal results from these units.

Automated hybridisation units may fail to provide sufficient humidity, leading to poor hybridisation. Try using humidified chambers, which can attain consistently-higher humidity levels.

Always balance the pH of wash solutions during preparation, and periodically during use, to ensure they produce optimal results.

Try rinsing slides in a dehydration series of EtOH washes (70%, 85%, and 100%) after the room temperature washes to reduce background.

When measuring the temperature of washing solutions, make sure to do this inside the jar to replicate the correct conditions.

Many types of fluorescent microscope filter degrade over time and should be treated as a consumable. Ensure regular maintenance and replace where necessary. 'Sputter' type filters are now available that last a lifetime.

Liquid light-guides in modern fluorescent light sources will degrade over time, resulting in reduced light transmission, and should be treated as a consumable. Make sure there are no sharp bends in the light-guide, schedule regular maintenance and replace when necessary.

Try to use just one type of immersion oil in your laboratory; different types of oil may be immiscible. Immersion oil of one type remaining on a microscope objective may result in a ‘milky’ appearance and a reduction of light transmission when it comes in contact with a slide with a different type of immersion oil.

Periodically washing solution jars and ensuring adequate detergent removal can help reduce background issues.

Having dedicated solution jars (e.g. Coplin jars only for hot wash) for processing steps can prevent poor results due to contamination with incompatible solutions.

Have you tried placing counterstained FFPE slides in a -20°C freezer for minimum of 20mins after FISHing? Some labs say this helps to produce sharper, more distinct probe signals.