Clear, bright FISH signals are only part of the equation.
Reducing background noise is just as important for achieving results that are easy to visualize and interpret.
If your signals are difficult to distinguish from the background, here are five areas to check before your next run.
1. Start With a Good Sample Foundation
Whether you’re working with a liquid suspension, FFPE tissue, or adherent cells, start with the cleanest sample possible.
What that means will vary by specimen type, but the goal is the same:
remove anything that could interfere with probe access or visualization. Debris, residual paraffin, extracellular proteins, and other contaminants can make analysis more difficult and contribute to unwanted background.
Teagan’s Tip: A cleaner starting sample can make the difference between searching for signals and seeing them clearly.
2. Clean Your Slides
Don’t overlook the slide itself. Residue or contamination on your slides can introduce fluorescence and background that make otherwise good signals harder to distinguish.
Make sure you’re using the appropriate slides for your application and that they’re
squeaky clean before you begin. Cleaning with ethanol or methanol, when appropriate for your workflow, can help eliminate unwanted residue.
Teagan’s Tip: Good FISH starts before the probe ever touches the slide.
3. Optimize Post-Wash Stringency
Post-hybridization washes play a major role in your
signal-to-noise ratio.
Too much stringency can reduce or eliminate your desired signal. Too little can leave nonspecifically bound probe behind, creating excessive background.
Finding the right balance helps preserve specific hybridization while removing unwanted fluorescence.
Teagan’s Tip: If you have weak signal or excessive background, your post-wash conditions are one of the first places to investigate.
4. Check Your Filters
It sounds simple, but your microscope can only display what its filters allow it to see.
Confirm that your filter set covers the appropriate
excitation and emission ranges for each fluorophore in your FISH probe.
You could have exceptionally bright fluorescent signals on your slide—but if your filter isn’t compatible with those wavelengths, you won’t see them properly.
Teagan’s Tip: Before troubleshooting the assay, make sure the problem isn’t your optics.
5. Use the Recommended Probe Ratio
More probe doesn’t necessarily mean more signal.
FISH probe concentrations and ratios are carefully optimized to balance signal intensity, specificity, and background. Using more—or less—than the recommended amount can produce less-than-ideal results.
Teagan’s Tip: Follow your probe provider’s Instructions for Use (IFU) and recommended probe volume before adjusting concentration.
Better Signal. Less Noise.
When FISH results aren’t as clear as expected, the solution doesn’t always require a major workflow change.
Sample preparation, clean slides, post-wash stringency, filter compatibility, and proper probe ratios can all have a significant impact on signal quality.
A few small adjustments may be all it takes to go from difficult-to-read fluorescence to
bright, distinct signals with a clean background.
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