by Rebecca Northeast, Technical Specialist, Proteintech Europe
Immunofluorescence is a popular antibody application that can suffer from high background due to tissue autofluorescence. This can be strong enough to mask the target signal and interfere with results, making it hard to differentiate between specific staining and noise in the slide.
What is autofluorescence?
Background fluorescence in a tissue that is not attributed to the specific staining of an antigen-antibody-fluorophore interaction is called autofluorescence. This arises from several causes, and the following steps will help you understand how to minimize their effects.
Tissue fixation with aldehyde cross-linkers
The most common fixatives are aldehyde cross-linkers such as 10% formalin or 4% paraformaldehyde (PFA). These work by creating protein-protein and protein-nucleic acid cross-links involving methylene bridges (-CH2-) which essentially glue the tissue together to form an insoluble meshwork, preserving tissue structure. In terms of fixatives that cause the most autofluorescence: glutaraldehyde is worse than paraformaldehyde, which is worse than formaldehyde.
Fixation-induced autofluorescence has a broad emission spectrum, occurring across the blue, green and red spectral range. Heating and dehydrating samples (commonly done in tissue IF) can also increase autofluorescence, causing greater emission in the red spectrum. One way to minimize this is to fix samples for the least amount of time required. Some papers have noted that treatment with sodium borohydride can help minimize these effects of fixation. If these troubleshooting options fail, then different fixatives such as chilled (-20 °C) ethanol or methanol are a good alternative for cell surface markers.
Figure 1. Endogenous autofluorescence of unstained formalin-fixed paraffin embedded (FFPE) kidney tissue taken using the green (left) and far-red (right) filters. Note how under the far-red filter the background autofluorescence is much less pronounced.
Autofluorescence from endogenous pigments
Autofluorescence can also occur due to native compounds in tissues. Examples include the heme group in red blood cells that contain the polyphyrin ring structure causing broad range autofluorescence. Perfusing with PBS prior to fixation is the best way to minimize this. Understandably, PBS perfusion is not always possible for some tissue samples such as post-mortem or embryonic tissue.
Other problematic endogenous pigments include collagen, NADH and lipofuscin. Collagen is a highly expressed, ubiquitous structural protein and has an emission spectrum in the blue region around 300-450 nm. NADH is an enzyme essential to many roles in metabolism in metabolically active cells and tissues, such as the liver, with an emission spectrum around 450 nm. Choosing a fluorophore with emission spectra in the red and far-red regions, such as CoraLite594 and CoraLite 647, will help distinguish specific staining from autofluorescence in tissues with high levels of compounds such as collagen and NADH (which emit in the blue/green spectrum). Another problematic compound is Lipofuscin which is a granular, lipophilic pigment that accumulates in lysosomes with age and fluoresces strongly at 500-695 nm. Sudan black B, a lipophilic dye, can effectively eliminate this autofluorescence.
Summary of autofluorescence troubleshooting
If autofluorescence is causing problems in your IF experiments, these general take-home points may hopefully resolve some issues and “un-shed” some light.
- Always perform endogenous tissue controls (no primary or secondary antibody) and primary antibody controls (just secondary antibody) to reveal the level of autofluorescence and non-specific binding in your immunofluorescence experiments.
- If possible, use alternative fixatives (such as organic solvents) or try paraformaldehyde over glutaraldehyde, and always fix for the minimum time required.
- Perfuse tissues with PBS prior to fixation to remove the presence of red blood cells.
- Use fluorophores that emit in a wavelength further from the autofluorescence compounds in your sample. Typically, far-red wavelength fluorophores such as CoralLite 647 are best for this.
- Sodium borohydride can be used (with mixed results) to decrease formalin induced autofluorescence.
- Lipofuscin and formalin autofluorescence can be reduced with reagents such as Sudan black B and Eriochrome black T.
About the Author: Rebecca Northeast is a Technical Specialist at Proteintech Europe. She provides application-specific talks and individual advice to increase engagement with the scientific community. Northeast also supports marketing and product management activities. She has a PhD in Neuroscience and a postdoc in Metabolism, as well as four first-author publications.
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