Immunofluorescence (IF) is a powerful but simple method for visualizing protein expression in tissue or cells using fluorophore-conjugated antibodies. But you need to optimize and understand the technique to balance your beautiful staining patterns with endogenous background noise. There’s nothing worse than looking under the microscope after a potentially day-long staining protocol following extensive tissue preparation, experimental treatment, etc., only to be greeted with high background noise or non-specific staining. One such source of background is autofluorescence due to structural tissue components, such as collagen and elastin or red blood cells, or as a result of aldehyde fixation, leading to broad emission across the tissue and obscuring your specific antigen staining. Recognizing this problem is crucial as it can interfere with your specific target staining, result in false positives, and introduce problems with assay validation and data credibility.
In this article, we will walk you through the process of recognizing an autofluorescence problem, outline some potential causes of autofluorescence, and teach you techniques for reducing or eliminating autofluorescence to uncover your beautiful, high-quality fluorescent staining.
What is autofluorescence?
Autofluorescence is background fluorescence due to naturally occurring substances which can affect your signal-to-noise ratio. It can arise through tissue fixation methods, especially when using aldehyde-based fixatives such as formalin, formaldehyde, or glutaraldehyde. It can also be inherent in the tissue. For example, red blood cells, collagen, and elastin would all be potential sources of autofluorescence. Another source is lipofuscin, which is an accumulation of proteins and lipids typically found in the brain and spinal cord in aged tissues. Even ink-based reagents, which can be effective for reducing red or green autofluorescence in lipofuscins, can introduce autofluorescence in the far-red end of the spectrum. No portion of the spectrum is safe from autofluorescence, so how do you know if you have it?
The short answer is if you turn on your laser and see almost uniform unexpected “signal” throughout your tissue that may be consistent across different channels, then you might be dealing with autofluorescence. Even when reducing exposure duration, autofluorescence might still be a problem, especially if your actual target signal is weak. An initial troubleshooting step might be to increase the concentration of either the primary or secondary antibody (or both!), but that can, at times, just lead to higher levels of background.
What are some ways to lower autofluorescence?
Typically, dyes have been deployed to reduce autofluorescence in some instances. As mentioned earlier, ink-based reagents, such as the hydrophobic dye Sudan Black B, can lower autofluorescence in the red and green channels by binding onto tissue and lowering fluorescence. However, Sudan Black B is not as effective at lowering autofluorescence due to aldehyde fixation or tissue elements, such as red blood cells and collagen. Similarly, other chemicals, such as copper sulfate and sodium borohydride, have varying degrees of success in reducing autofluorescence in some cases, but not others. Why waste precious tissue and time on inferior methods for reducing autofluorescence when you can reveal true immunofluorescent staining in a consistent manner?
How can you say goodbye to autofluorescence?
Vector Laboratories offers TrueVIEW® Autofluorescence Quenching Kits to dramatically reduce non-lipofuscin autofluorescence from tissue elements, red blood cells, or aldehyde-based fixatives. The hydrophilic, nonfluorescent molecule in the kit binds electrostatically to collagen, red blood cells, and elastin, as well as aldehyde-fixed tissue, to significantly reduce autofluorescence. Treatment with this kit requires just 5 extra minutes at room temperature towards the end of your immunofluorescence protocol and is compatible with many common fluorophores such as GFP, AlexaFluor®, FITC, DyLight®, and cyanines. It even works in problematic tissue types such as kidney, spleen, and pancreas.
The kit also includes an antifade mounting medium with the flexibility to use a DAPI counterstain. In this three-step protocol, all you need to do is mix the three reagents in a 1:1:1 ratio, apply the working solution to your tissue, then coverslip and visualize. It’s that easy. Now you can increase exposure time, bringing out your target of interest with a strong intensity worthy of your LinkedIn cover photo.
How it works
Explore the simple workflow. Say goodbye to unwanted autofluorescence, while dramatically improving signal-to-noise ratio. If you’re ready to get a “true view” of your intended target, be sure to use this kit! Check out the app note or watch our webinar to learn more.
Explore the simple workflow that will let you say goodbye to unwanted autofluorescence, while dramatically improving signal-to-noise ratio. Reveal true immunofluorescence with the Vector® TrueVIEW® Autofluorescence Quenching Kit.