Troubleshooting Autofluorescence in Your Samples

Sample autofluorescence can be one of the trickiest aspects of working with fluorescent imaging. Although HCR™ Gold and HCR™ Pro have been engineered to generate strong signal amplification to overcome this property inherent to all tissue samples, there can certainly be cases that autofluorescence presents a challenge to image analysis or interpretation. Here are some possible approaches for mitigating sample autofluorescence. While the variable nature of autofluorescence makes it difficult to have a one-size-fits-all solution, these may be incorporated into your own workflow as starting points.

1. Strategic Channel Selection
   • Autofluorescence is generally the highest in shorter wavelength channels (particularly green/488), so using red or far-red channels (594, 647, 700) for lower abundance targets can help avoid unwanted interference.
   • Note: certain tissue types, such as those with high abundance of red blood cells, can have unique autofluorescence signatures in other fluorescent channels also.
2. Chemical Bleaching
   • ‍3% H₂0₂ and 20 nM NaOH in PBS. This is a peroxide-based solution that can be readily made in labs and has been shown to help lower sample autofluorescence. The sample incubation time for optimal bleaching may vary depending on your specific sample/tissue. Following incubation, wash the sample 3 times in PBS before moving on to the appropriate HCR™ protocol.
   • Note: to strengthen the treatment, increase incubation time or concentration of peroxide to 4.5% H₂0₂ and 24 nM NaOH in PBS.
3. Photobleaching
   • Photobleaching works by reducing autofluorescence by light-based bleaching prior to the HCR™ protocol. Similar to chemical bleaching, the optimal type of photobleaching can vary based on your sample/tissue. You can try incorporating pre-exposure to strong broadband visible light or UV light.
   • Note: photobleaching can be combined with the aforementioned chemical bleaching for stronger effects.
4. Commercially-Available Reagents
   • Several commercially available reagents have been shown to reduce sample autofluorescence, including: ‍
     1. TrueBlack‍‍
     2. Sudan Black
   • Note: These reagents should be applied after the HCR™ protocol.
   • For optimal results, we suggest reaching out to the manufacturers for guidance.

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