The fluorescence present in many tissues due to endogenous compounds, termed autofluorescence, introduces a confounding factor for techniques such as fluorescence immunohistochemistry or fluorescence in situ hybridization. If the emitted light from the autofluorescence overlaps spectrally and spatially with the introduced fluorophore, distinguishing the two is difficult if not impossible using standard widefield or confocal microscopy. Techniques such as spectral unmixing or fluorescence lifetime imaging microscopy can be used to temporally or computationally remove autofluorescent signals, but they are only available on highly specialized microscope systems.
Although numerous endogenous compounds exhibit fluorescent properties to varying degrees, the substance lipofuscin is responsible for a substantial amount of autofluorescence in a number of tissues, including cardiac muscle, smooth muscle, and especially brain. Lipofuscin, sometimes called “the aging pigment,” is a highly complex assembly of lipids, proteins, metals, and other unknown components. It accumulates in long-lived cells, such as cardiac myocytes or neurons, and fluoresces brightly over a wide spectral range (excitation: 350-580 nm; emission: 400-630 nm).
Recently, Chemicon International introduced an Autofluorescence Eliminator Reagent (Cat. #2160), enabling the reduction of autofluorescence due to lipofuscin. The reagent consists of a solution of Sudan Black (and possibly other components) in ethanol and is supplied in a medium-sized, opaque dropper bottle. The effectiveness of autofluorescence reduction was tested in our laboratory on adult human brain tissue following the manufacture’s instructions. Lipofuscin is particularly abundant in neurons in the adult human brain. Free-floating sections were stained using a primary antibody against the neuronal enzyme choline acetyltransferase and visualized with a Cy3-conjugated secondary antibody. After washing, they were placed in 70% ethanol for 5 min followed by incubation in the Autofluorescence Eliminator Reagent for 5 min. This was followed by three changes in 70% ethanol for 1 min each before mounting in glycerol. The sections were examined with a confocal microscope using a 543 nm laser line for excitation and a 555-625 bandpass filter for emission. Without treatment, lipofuscin-containing neurons exhibit a high level of autofluorescence. This autofluorescence is reduced significantly following treatment with the Autofluorescence Eliminator Reagent. Immunofluorescent staining performed using Cy3 before treatment (as recommended) is reduced marginally (5-10%) after treatment.
On balance, the reagent performed well. It is simple to use and does significantly reduce autofluorescence due to lipofuscin. The portion used can be saved and reused multiple times. The reagent would be useful to any researcher experiencing unwanted fluorescence due to lipofuscin, especially those working with primate brain tissue, including that from humans. The only drawback is a small reduction in the fluorescent signal from the introduced fluorophore (in this case, Cy3). It also must be kept in mind that the reagent is not thought to reduce autofluorescence from non-lipofuscin sources, such elastin, collagen, chlorophyll, or glutaraldehyde fixation.
Michael Hendrickson, B.S.
Research Assistant
W.M. Keck Laboratory for Biological Imaging
University of Wisconsin-Madison