March 1, 2018 (Vol. 38, No. 5)
Timothy Karpishin Ph.D. Director of Chemistry Vector Laboratories
Innovations in Immunofluorescence Analysis Boost Signal-to-Noise Ratios
Immunofluorescence is a common technique in modern biology and medicine. It is a highly sensitive method used primarily for the analysis of tissue samples with a fluorescence microscope. When this technique is used, antibodies are labeled with a fluorescent molecule to localize the antigen–antibody complex. Immunofluorescence can provide clear subcellular visualization of proteins, glycans, and small biological and nonbiological molecules. However, tissue autofluorescence often impairs or prevents the effective use of this tool. As will be described, a new technology has been developed that significantly lowers autofluorescence and dramatically enhances signal-to-noise ratios.
Autofluorescence is a general term describing the background fluorescence in tissue sections unrelated to the specific signal generated during an immunofluorescent assay. Tissue components such as red blood cells (RBCs) and collagen are strongly fluorescent, making it difficult to discern relevant signal from background noise. In addition, formalin fixation, which is commonly used to immobilize antigens while retaining cellular structure, introduces a significant amount of fluorescence as well.
Background autofluorescence makes the interpretation of assay results particularly troublesome with green- and red-channel fluorophores. Although long-wavelength (far-red) fluorophores were developed in part to address this problem, background autofluorescence can still occur in the 600–700 nm range in some cases.
Tissue autofluorescence is often due to components native to tissue. These include flavins, porphyrins, chlorophyll (in plants), collagen, elastin, RBCs, and lipofuscin. These components are generally fluorescent in the green and yellow portions of the visible spectrum. The most commonly used fluorophores in immunofluorescence assays are the green channel fluorophores fluorescein and Alexa Fluor 488 (Thermo Fisher Scientific), and natural tissue components can therefore significantly affect the signal-to-noise ratio when these fluorophores are used.
Autofluorescence also results from the use of aldehyde fixation (formaldehyde or glutaraldehyde). Many formalin-fixed paraffin-embedded (FFPE) tissue samples, specifically FFPE kidney and spleen, are unsuitable in immunofluorescent assays due to high background fluorescence. Fluorescence due to formalin fixation leads to broad emission over a wide spectral range including blue, green, and red emission.
The mechanistic features of tissue fixation that lead to a significant increase in fluorescence are not well understood. Both formaldehyde and glutaraldehyde stabilize tissue by forming covalent crosslinks. The chemical structures of the crosslinks are diverse due to the variety of protein side chains that react with the aldehyde, the slow kinetics and reversibility of the reactions, and the complex aqueous chemistry of the crosslinking reagents.
Established Means of Lowering Autofluorescence
Nonetheless, it is expected that the increase in fluorescence seen in FFPE tissue is due to the generation of C=C or C=N bonds. To address this problem, sodium borohydride has been utilized in several studies to chemically reduce the C=C and C=N bonds and diminish the autofluorescence. Borohydride and similar reducing agents, however, are unstable at neutral pH. Whereas researchers have noted some success when using sodium borohydride to lower tissue autofluorescence, the procedure is not reproducible and the reagent is difficult to work with.
An additional method that has been investigated to diminish tissue autofluorescence is photobleaching. When this technique is used, tissue sections are exposed to high-intensity UV radiation for long periods of time to irreversibly photo-oxidize the fluorescent tissue elements. Photobleaching, which is often used in conjunction with other treatments, has been shown to be somewhat effective; however, it is time consuming. Also, photobleaching requires equipment that most immunohistochemistry laboratories lack.
Historically, the main method that has been employed to lower tissue autofluorescence has been to treat the tissue with solutions of Sudan Black or similar nonfluorescent diazo dyes. These hydrophobic dye molecules will generally bind nonspecifically to tissue sections. After binding to the tissue, Sudan Black acts as a mask to lower the fluorescence through the absorption of incident radiation (dark quenching).
Specifically, Sudan Black is very effective at lowering the fluorescence due to lipofuscin in tissue. Lipofuscin is a brightly fluorescent pigment that accumulates with age in several tissue types. Lipofuscin granules are composed of lipid-containing residues of lysosomal digestion. Because of the hydrophobic nature of Sudan Black, it binds effectively to the lipid-rich lipofuscin granules and effectively masks their fluorescence. However, Sudan Black is less effective at lowering the fluorescence due to aldehyde fixation, connective tissue elements, and RBCs.
A new method has been introduced that allows for the dramatic reduction of the autofluorescence due to formalin fixation, collagen, elastin, and RBCs. This innovative technology, sold as the Vector TrueVIEW Autofluorescence Quenching Kit, involves the treatment of tissue sections with an aqueous solution of a hydrophilic molecule that binds electrostatically to collagen, elastin, and RBCs. This nonfluorescent, negatively charged molecule also binds effectively to formalin-fixed tissue including colon, pancreas, prostate, tonsil, spleen, kidney, gallbladder, and thymus.
Once bound, the TrueVIEW Quencher significantly lowers the fluorescence of tissue components. It is likely that this reagent lowers autofluorescence through a combination of static and dark quenching mechanisms.
Because the TrueVIEW Quencher is hydrophilic, the treatment of tissue sections is performed in an aqueous buffer, and does not require a 70% ethanol pretreatment step that is necessary with Sudan Black procedures. In addition, the TrueVIEW Quencher treatment requires only a two-minute step at the end of the immunofluorescence assay. Further, TrueVIEW Quencher is compatible with common fluorophores such as fluorescein, Alexa Fluors, DyLight fluors, cyanine fluors, and green fluorescent protein.
The usefulness of TrueVIEW Quencher treatment has been demonstrated in an analysis of spleen tissue (Figure 1A & 1B) and pancreas tissue (Figure 1C & 1D). Prior to treatment of spleen tissue, considerable autofluorescence occurs in the green channel and obfuscates the signal due to Ki67 (Figure 1A). After treatment, a clear representation of Ki67 targets is seen as bright green punctate staining on a black background (Figure 1B). Figure 1 also demonstrates the effect of using TrueVIEW Quencher with pancreas tissue, where both green and red background autofluorescence is eliminated with treatment (Figure 1C & 1D). Figure 2 demonstrates the diminishment of green and red autofluorescence in kidney tissue, and it shows the preservation of DAPI nuclear staining visible in the blue channel.
The primary concern with any attempt to lower tissue autofluorescence is the effect of the treatment on the desired signal (usually a fluorophore-linked secondary antibody). In an ideal situation, the autofluorescence would be eliminated with no effect on the signal of the fluorescent secondary antibody.
With the use of TrueVIEW Quencher, there is substantial lowering of background autofluorescence with only a modest loss in the brightness due to the fluorescent secondary. This effect can easily be compensated for by the addition of a higher concentration of the primary antibody (Figure 3), or by increasing the camera exposure time when obtaining digital images. With these considerations, the use of Vector TrueVIEW Autofluorescence Quenching reagent leads to a significant enhancement in overall signal-to-noise ratio in most immunofluorescent assays.
For the expanding field of immunofluorescence, a new method has now been developed that is rapid, is easy to use, and allows the researcher to use FFPE specimens in investigations that were previously unacceptable due to autofluorescence. This method has broad application for tissue-based immunofluorescence assays and is compatible with standard fluorescence and confocal laser microscopes.