How do I reduce high background in my FISH assay?

As a leading supplier of fluorescence in situ hybridisation (FISH) probes and cytogenetic resources for over 20 years, it’s fair to say that we’ve seen our fair share of benchtop experience here at OGT. Among the various technical challenges that our partners encounter, one of the most common is the issue of high background fluorescence in FISH assays.

High background fluorescence is more than just a nuisance – it’s a significant issue that obscures critical data, complicates interpretation and may result in erroneous conclusions. The sources of high background are varied and can stem from multiple aspects of the FISH procedure, from denaturation temperature to washing protocols.

In this blog, we’ll delve into the key factors contributing to high background in FISH assays and provide actionable strategies to help you achieve the crystal-clear results you need. Whether you’re troubleshooting your current protocol or looking to refine your technique, this guide will help you enhance the accuracy and reliability of your FISH analyses.

Refine your sample preparation technique

Proper preparation of test samples is one of the most crucial steps in achieving high-quality FISH results. The preparation process lays the foundation for the entire assay, and even minor deviations can lead to significant issues such as uneven probe binding and elevated background fluorescence.

Fixation of FFPE samples is particularly critical because it preserves the cellular architecture while maintaining the accessibility of the target DNA sequences. The fixation process is a delicate balance; both under-fixation and over-fixation can lead to a high background signal.

• Under-fixation can result in incomplete preservation of the cellular structure. This increases the risk of DNA degradation and non-specific binding of the probe, often leading to high background fluorescence.
• Over-fixation with formalin can prompt excessive cross-linking of proteins and nucleic acids. This can compromise cell permeability and mask target sequences, making them less accessible to the probes. This not only reduces the overall signal intensity, but also elevates background signal through increased non-specific binding.

Our top tips

For best results, always use freshly prepared fixative solutions and adhere to the fixation times stated in the protocol closely. If using Carnoy’s solution for cell fixation, always use freshly prepared solution, stored at -20°C to prevent moisture absorption and maintain its effectiveness, discard the solution after use.

For blood smear slides, hypotonic solutions e.g. potassium chloride can be used during fixation to reduce background fluorescence. When using formalin-fixed paraffin-embedded (FFPE) tissue, we suggest aiming for sections of 3-4μm thick to avoid issues with probe penetration and interpretation.

For more tips on FISH sample preparation, visit our Sample Preparation FAQs

Take extra care during pre-treatment steps

Pre-treatment assay steps, such as enzyme digestion or heat treatment, can also be a source of high background. Pre-treatment measures are often employed in FISH assays to break down proteins, lipids and other cellular components that may mask or obstruct the target DNA sequences.

• Insufficient pre-treatment may leave behind cellular debris that exert natural autofluorescence, or that may act as nonspecific binding sites, where the probe binds to non-target sequences, prompting high background signal.
• Over digestion may damage your sample and target sequence, resulting in low signal.

Figure 1. Example FISH images of (A) Under-fixation of a blood smear, (B) Insufficient pre-treatment of FFPE tissue, (C) Under-digestion of FFPE tissue and (D) Over-digestion of FFPE tissue

Our top tips

For optimal results on your FFPE tissue samples we suggest you use the CytoCell^® LPS 100 Tissue Pretreatment Kit. When using this kit, make sure to heat the Tissue Pretreatment Solution (Reagent 1) to 98–100°C in a water bath and maintain this temperature when you introduce your slides for at least 30 minutes (this may increase depending upon your tissue type, fixation and/or section size) before treating with enzyme at 37°C. Remember to refresh your pre-treatment solution between slide batches.

Optimise your probe volume and denaturation conditions

Probe volume is key in determining the success of your FISH assay, influencing the specificity and clarity of your results. Using an optimal probe volume helps to maximise specific binding between the probe and target sequence, ensuring strong signal at the target while minimising background fluorescence.

Another important factor to consider, particularly when using FFPE tissue, is the denaturation conditions. During this step, the double-stranded DNA is unwound into single strands, allowing the probe to bind to its complementary target sequence. For FFPE samples, which are highly cross-linked following formalin fixation, using the optimal denaturation temperature and time, as stated in your FISH probe protocol, is critical to ensure probe hybridisation without increasing background fluorescence.

• Probe volume: Conversely, too low a probe volume can result in weak signals, making it difficult to detect the target sequence.
• Denaturation temperature is a crucial parameter that must be carefully optimised and maintained. Too low a temperature can prevent effective probe binding, while too high a temperature may give rise to non-specific binding, both of which can compromise the clarity of your results.
• Denaturation time also plays a role in background levels. Short denaturation times might not allow sufficient unwinding of the FISH probe and target DNA double strands resulting in reduced binding of the probe to the target sequence ultimately producing weak signals. However, prolonged denaturation times can increase unmasking of non-specific binding sites allowing for off-target binding of FISH probes thus contributing to increased background signals.

Having issues with high background in your haematology FISH assay? Check out our specialist haematology FISH troubleshooting resource

Master your washing steps

Effective washing is a critical component of FISH assays, reducing background fluorescence by removing excess unbound or non-specifically bound probes. Washing steps must be carefully controlled to ensure that non-specifically bound probes are thoroughly removed without affecting the specific probe-target hybrids.

• Stringency washes: optimising the stringency of your washes is crucial for reducing non-specific binding. High stringency washes help remove probes that have bound to non-target sequences, thus lowering background noise. However, be cautious; too much stringency can lead to the loss of specific signals, while too little stringency will fail to remove enough background.
• Wash buffer quality: The quality and freshness of your wash buffers are equally important. Degraded or contaminated buffers can fail to effectively remove non-specifically bound probes, or at worst, may introduce unwanted background fluorescence.

Our top tips

To minimise background fluorescence, optimise the stringency of your washes by carefully adjusting your incubation pH, temperature and time. Begin with the recommended incubation time in the protocol and make incremental changes if background persists. Always use freshly prepared wash buffers to prevent contamination or degradation.

Learn more about the importance of proper reagent storage and preparation in our FISH reagent storage guide

Check your optical filters

You might have performed your FISH assay perfectly up until now, but there remains one more obstacle that could scupper your success. The optical filter in your fluorescence microscope can be another source of weak overall signal and cloud your results.

• Worn or damaged filters will exhibit a mottled appearance as they degrade, which if left alone, will eventually completely obscure the filter. The effects may not be noticeable on a day-to-day basis but will manifest in weaker signals.

Figure 2. An example of a damaged filter

Our top tips

Check your optical filters for signs of wear or damage. Extend the useful life of your optical filters by protecting them from the light source as much as possible. One way to achieve this is by closing the microscope shutter when not in use.  Replace filters in line with manufacturer’s guidelines – typically every 2-4 years.

Wave goodbye to high background fluorescence in your FISH assays

High background fluorescence is the enemy of strong FISH assay results and can be influenced by a myriad of assay parameters. By refining your sample preparation techniques, pre-treatment steps, probe volume, denaturation conditions and washes, you can significantly enhance the clarity of your FISH results and be confident in your analysis and research findings.

And so, remember to help reduce high background fluorescence:

• Where possible, minimise probe exposure to light
• Always use freshly prepared fixative solutions
• Consider using hypotonic solutions during blood smear fixation to reduce background fluorescence
• Aim for FFPE sections of 3-4μm thick to avoid issues with probe penetration and interpretation
• Use the CytoCell LPS 100 Tissue Pretreatment Kit for optimal results on your FFPE tissue samples
• Always use freshly prepared wash buffers to prevent contamination or degradation
• Check your optical filters for signs of wear or damage and replace filters in line with manufacturer’s guidelines

OGT’s ready-to-use CytoCell FISH probes come with pre-optimised protocols that can help you solve the issues we’ve covered here, explore our comprehensive range of FISH probes and ancillary materials here

Still struggling with FISH assay troubleshooting? Reach out to us today to enquire about a FISH health check with our team of expert Field Application Specialists