Tau IF Background: Causes, Culprits, and How to Fix It

When performing an immunofluorescence (IF) experiment, time and samples are often quite valuable. This can especially be the case for tissue from the central nervous system (CNS), which is often used to study the microtubule-binding protein tau. Researchers often use antibodies to detect tau in tissue to gain insight into how this protein becomes dysregulated and spreads in a prion-like manner from cell to cell. However, high background during IF can pose a threat to the next breakthrough in neurodegeneration-related tau research.

Background during IF experiments can take several different forms. This can look like a colorful haze across images, speckles of color throughout a sample, and nonspecific staining of every protein within a given sample. While it might be easy to rule out a hazy background, the prevalence of tau in neurons and other CNS cells might make it difficult to diagnose nonspecific staining as background versus what one would hope to see in an experiment targeting tau. Even more, nondescript background speckles threaten to deceive researchers who are trying to hone in on the precise mechanism of how tau aggregation spreads from cell to cell. With neurodegeneration and tauopathies increasingly affecting our aging populations, time shouldn’t be wasted on hypotheses based on background noise.

What could be contributing to high IF background during a tau experiment?

Typically, the biggest contributor to nonspecific background during IF comes from the antibodies that are being used to detect a target. While the typical range for antibody dilution during IF lies between 1:100 and 1:1000, increased antibody concentration risks greater nonspecific background.

For this reason, it is essential for researchers to use antibodies that are validated at a range of dilution levels that do not require such high concentrations. Currently, HUABIO offers three IF-validated tau antibodies, including antibodies for phospho-tau Ser396, which is validated for use as low as 1:500. HUABIO's tau antibodies are all validated for IF in cells at dilutions up to 1:500, which is suitable for the balancing act of detecting the highly abundant tau protein in CNS-derived cells against the potential for background attributable to the concentration of primary antibody.

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Other factors that could influence IF background when studying tau are blocking and washing, which should be optimized per each application. Likewise, secondary antibodies can also contribute to nonspecific background, so secondary-only controls should always be run during an IF experiment.

Curiously, one particular case that may be observed for background during experiments that target tau in brain-derived tissue is the presence of a pigment called lipofuscin. Lipofuscin is an autofluorescent type of granule that results from the unique longevity of neurons and other brain cells. While other cells are continually dividing and clear cellular waste through normal turnover, neurons are long-lived and do not divide, resulting in the buildup of cellular trash. This cellular trash goes into the lipofuscin granules, which produce an autofluorescent yellow-green signal. This specific autofluorescence is quite evident in the types of tissues often studied as models for understanding neurodegeneration and tau, so researchers new to brain tissue IF should be aware of its potential to cause background signal. Fortunately, there are special chemical reagents that can be added to brain tissue that specifically reduce the background contributed by lipofuscin. Between these reagents and having a high quality tau antibody, your next IF experiment should be ready to target biological phenomena without any confusion. 

Not sure which antibody to use for your tau IF experiment? Want to stain or probe tau with other experimental protocols? Feel free to reach out with technical questions and troubleshooting to our human-monitored support inbox at [email protected].