The human brain autofluoresces—a funny thought next time you see a cartoon character with a bright idea and a light bulb over his head—but not so funny if you are attempting immunofluorescence analysis. But there are some significant advantages to using fluorescence detection over chromogenic methods. In this article, I will cover the advantages of fluorescent imaging and how to minimize autofluorescence in your brain sample, so you can take advantage of the best techniques.
Autofluorescence also occurs in the brains of rodent animal models used in research, but to a much lesser extent than in human brains. And in animals, fluorescent antibody signals should usually be much brighter than the intrinsic autofluorescence. But with human brain samples, the autofluorescence is so significant that most researchers use chromogenic immunohistochemistry methods, such as HRP/DAB reactions, specifically to avoid it. But it doesn’t have to be.
Why Fluorescence Immunohistochemistry Rocks
1) You can Detect Multiple Antigens
With chromogenic immunohistochemistry, you are usually limited to one- or two-color detection. Chromogenic detection uses a chemical reaction between your secondary antibody and a visualizing agent to cause a stain to deposit onto your sample. Then the stain is detected by eye or by a microscope camera. This limits you to usually two colors.
With fluorescence, you can simultaneously localize two, three, or even four proteins of interest. This is because fluorescent secondary antibodies have unique excitation and emission wavelengths. So you can use filter cubes or tunable lasers to shine light of a specific wavelength on your sample and excite just one fluorophore at a time. Then based on the known excitation and emission profiles of the fluorophores coupled to the secondary antibodies, you can distinguish numerous antigens from one another.
2) You can Examine Dyes and Fluorophores in the Same Section
In my field, researchers study brain protein aggregation disorders such as Parkinson’s Disease (PD) and Alzheimer’s Disease (AD). The aggregates in these diseases are often assessed by fluorescent amyloid-binding dyes, such as Congo Red or Thioflavin S, which respectively bind amyloid plaques (in AD) and Lewy Bodies (in PD). But due to overlap between the dye’s imaging properties and chromogenic detection, researchers are often forced to do two experiments on adjacent serial sections:
- Fluorescent detection with dyes, and
- Colorimetric immunohistochemistry.
There is a problem with looking at serial sections instead of both elements stained in the same section, you can not directly compare regions of the protein aggregate that bind the dye versus those that contain your protein of interest. This is a good reason to use immunofluorescence instead of chromogenic imaging. If you use fluorescent antibodies against post-translational modifications of a protein in the aggregate you can directly compare it to the signals from an amyloid binding dye. Then you can actually determine if your protein modification is associated with the amyloid conformation.
How to Manage Autoflourescence
Before we talk about how to get rid of autofluorescence, let’s talk about where it comes from: lipofuscin. Pigmented lipofuscin granules are a natural byproduct of lysosomal activity in the cell. Interesting fact: lipofuscin is actually considered an aging pigment, since its presence is increased with age in many organs in the body. In fact, lipofuscin is so well correlated with age that it’s used to calculate the age of crustacean animals such as lobsters.
Here are three reasons why this type of autofluorescence is hard to work around:
- It has very broad excitation and emission spectra—it will show up in a number of your fluorescence channels. This means you can’t simply accept that it will take up your blue channel, for example, and use the other channels for your antibodies or dyes.
- It is very difficult to photobleach. Some reports say that you can irradiate your tissue for a few days before staining, but this not always sufficient to completely reduce the autofluorescence. Importantly, this process can easily cause tissue damage.
- Lipofuscin granules tend to cluster around the nucleus of nerve cells, which is often where we find Lewy Bodies in neurodegenerative diseases. This spatial overlap makes it hard to separate Lewy Body staining from the autofluorescent background.
So, how do you overcome this wild and wacky autofluorescence that’s always on in your brain (and, more importantly, the human brain samples you’re working with)? There are two tricks that I can share—one optical, one chemical.
The Chemical Trick
The chemical trick involves quenching the autofluorescence using a variety of homemade or commercially available quenchers. Sudan Black is the most popular and reliable. Chemical quenchers are applied after antibody or dye staining and often require harsh pre-treatment and washing conditions that can affect tissue morphology. The main drawback with using a chemical method is you may modify, reduce, or even cover up your desired staining. Since the quenching happens after your immunohistochemistry procedure, it’s hard to know if quenching has changed the staining pattern. Not a great option.
The Optical Trick
The other trick for overcoming autofluorescence is to optically isolate the autofluorescent signal and subtract it from your image. To do this you need access to specialized laser scanning confocal microscopes and software that are capable of spectral imaging with linear unmixing. These systems can separate the light coming off your sample into ~10 nm wavelength segments by dividing the emission light into its individual spectral components. If you know the distinct emission profiles of your autofluorescence, dye labeling, and fluorescent secondaries, you can then separate these signals even if they are very close to each other in the same tissue section.
- Controls are essential to any experiment. For fluorescence detection be sure to have an unstained sample. This is needed so you can clearly measure the different autofluorescent components of the tissue—without your antibody or dye signal getting in the way. Also include single color samples for each of your dyes or secondary antibodies. You will use these to determine the individual profiles of each fluorescent component in your sample.
- Photobleaching becomes a bigger issue in immunofluorescence compared to colorimetric imaging. Always try to use the lowest intensity of light that you can get away with while still being able to see your signal. You can assess the amount of photobleaching by checking the signal intensity at a new region of your sample that you haven’t yet imaged.
Given the advantages of using fluorescent immunohistochemistry for human brain samples, it is worth the time and effort to recognize and work around autofluorescence. If you’ve worked with these or other methods to eliminate autofluorescence, share with us your experiences, tips, and tricks in the comments section.