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FISH 'n' Tips

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We’re gathering information from our Cytocell experts to share the latest and most helpful tips for FISH. Visit this page each month for new, timely, proven tips from the FISH experts. Choose from the sections listed below to begin viewing the related tips and tricks.

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New Tip! 

Featured tip example

During FISH reagent storage and disposal, when not in use, ensure that the enzyme solution is stored correctly at 2-8°C to preserve the enzyme activity.

View FISH 'n' Tips for:

  1. Reagent storage and disposal
  2. Sample preparation
  3. Probe application
  4. Denaturation
  5. Hybridization
  6. Post hybridization washes
  7. Analysis
  8. General tips

1. Tips for reagent storage and disposal

Section 1 Reagent Storage and Disposal

  • NEW TIP

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

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2. Tips for sample preparation

Section 2 Preperation

  • 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.
  • 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.
  • 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.
  • Consider using a mild hypotonic treatment before fixing CD138+ selected cells, which may improve cell morphology.
  • Positively charged slides should only be used for FFPE sections. If these slides are used for cell suspensions, they may produce high green background.
  • 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.
  • When using archived cell pellets, consider re-fixing using fresh fixative to ensure they provide optimal results.
  • Consider using Pepsin to digest excess cytoplasm and cellular debris in Cytospin samples to improve hybridization quality.
  • Try cleaning your slides in 70% ethanol before use, to get rid of any dust or debris. A high degree of debris may lead to a high background.

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3. Tips for probe application

Section 3 Probe application

  • 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 hybridization.
  • To avoid patchy hybridizations, 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.
  • Try cleaning your slides in 70% ethanol before use, to get rid of any dust or debris. A high degree of debris may lead to a high background.

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4. Tips for denaturation

Section 4 Denaturation v2

  • Use calibrated temperature-measuring devices to regularly QC hybridization 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 hybridization units, to ensure they are within manufacturer specifications.

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5. Tips for hybridisation

Section 5 Hybridisation

  • Test ramp up and ramp down temperature times periodically as part of equipment QC on automated hybridization units, to ensure they are within manufacturer specifications.
  • Maintaining humidity strips on automated hybridization units according to manufacturer recommendations can help ensure optimal results from these units.
  • Automated hybridization units may fail to provide sufficient humidity, leading to poor hybridisation. Try using humidified chambers, which can attain consistently-higher humidity levels.

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6. Tips for post hybridisation washes

Section 6 Post hybridisation washes v4

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

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7. Tips for FISH probe analysis

Section 7 Analysis

  • 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.

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8. General tips

Section 8 FISH general

  • 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.

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