Question: What steps can I take to minimize the impact of autofluorescence during immunofluorescence staining?
Answer: Autofluorescence can result from aldehyde fixation leading to high background fluorescence in a wide spectral range, including blue, green, and red emissions.
It's important to know the biology of the tissue of interest. Understanding the tissue elements, cells, and structures that exhibit autofluorescence will give you the signal intensity of autofluorescence in different wavelengths. Below are general steps for assessing autofluorescence:
- Stain and image your tissue of interest with the primary antibody omitted. If the tissue elements that contribute to autofluorescence don't interfere with your specific signal, they can be ignored.
- If the autofluorescence does interfere and your specific signal is stronger than the autofluorescence, reduce the exposure time until the signal is visible and autofluorescence is weaker or not visible.
- If the autofluorescence does interfere and it's stronger than the specific signal of your antibody, use a fluorophore outside of the emission spectra of the autofluorescence; such as far-red/near-infrared fluorescent dyes (e.g., Alexa 647 or 750).
- Usually, autofluorescence is stronger in the green emission spectra and weaker in infrared emission spectra. Therefore putting the antibody that gives the strongest signal in green emission spectra can help to minimize the impact of autofluorescence.
- Using narrow band emission filters or spectral unmixing can help to remove the autofluorescence of your image. Strive to optimize a protocol where you can obtain the strongest possible signal especially with green and red fluorophores. These optimizations can be done using SuperFrost or ColorFrost Slides.
- If you are still not happy with the autofluorescence and it interferes with your signal (even after optimization), you can test out quenchers.
From the limited testing we have performed with the quencher TrueBlack, we did not observe any significant difference in assay performance (e.g. Median UMI Counts per Spot, Median Genes per Spot, or clustering) when compared to tissues untreated. Therefore, it's likely low risk to use TrueBlack for your immunofluorescence staining with Visium for FFPE.
When you use TrueBlack be sure it does not quench your signal. This can happen when the signal is low. When the signal is strong you may not need to use a quencher.
Product: Visium for FFPE