ARTICLE |
Correspondence to: William E. Klunk, Dept. of Psychiatry, U. of Pittsburgh, 705 Parran HallGSPH, 130 DeSoto Street, Pittsburgh, PA 15213-2535. E-mail: klunkwe@msx.upmc.edu
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Summary |
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X-34, a lipophilic, highly fluorescent derivative of Congo red, was examined as a histochemical stain for pathological changes in Alzheimer's disease (AD). X-34 intensely stained neuritic and diffuse plaques, neurofibrillary tangles (NFTs), neuropil threads, and cerebrovascular amyloid. Comparison to standard methods of demonstrating AD pathology showed that X-34 correlated well with Bielschowsky and thioflavin-S staining. X-34 staining of NFTs correlated closely with anti-TAU antibody staining. A 1:1 correspondence of X-34 and anti-Aß antibody staining of plaques and cerebrovascular amyloid was observed. Both X-34 and thioflavin-S staining were eliminated by formic acid pretreatment, suggesting that ß-sheet secondary protein structure is a necessary determinant of staining. X-34 may be a general amyloid stain, like Congo red, because it also stains systemic amyloid deposits due to -light chain monoclonal gammopathy. In conclusion, X-34 is a highly fluorescent marker for ß-sheet structures and intensely labels amyloid plaques, NFTs, neuropil threads, and vascular amyloid in AD brains. It can be used with both paraffin-embedded and frozen tissues as well as in combination with immunohistochemistry for double labeling. The intensity of staining and the simplicity and reproducibility of the technique suggest that it may be a useful addition to the standard techniques for evaluation of AD neuropathology. (J Histochem Cytochem 48:12231232, 2000)
Key Words: Alzheimer's disease, plaques, neurofibrillary tangles, neuropil threads, fluorescence microscopy, X-34, thioflavin-S
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Introduction |
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CONGO RED has been used for decades to identify amyloid deposits in post-mortem tissue (
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This study explores the properties of X-34 compared to standard staining methods used in the study of AD neuropathology, including antibodies to Aß and TAU protein, Bielschowsky silver stain, and thioflavin-S. In addition, we define the optimal staining conditions, including optimal staining concentrations and staining times and optimal filter sets for fluorescence microscopy. Finally, we explore the specificity of this dye for ß-sheet fibrils.
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Materials and Methods |
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Synthesis of X-34
X-34 was synthesized using standard organic chemistry techniques through a HornerEmmons reaction (
Tissue
Postmortem brain tissue from 12 autopsy-confirmed AD patients and six cognitively normal controls was obtained through the University of Pittsburgh Alzheimer's Disease Research Center Brain Bank. Age at death of AD patients was 71.3 ± 2.7 years (range 6975) and that of controls was 77.8 ± 6.3 years (range 7085). The postmortem interval was 4.0 ± 1.3 hr (range 25) for AD brains and 7.1 ± 3.7 hr (range 312) for controls. Frontal cortex was dissected from 1-cm-thick coronal slices and was placed in either 10% buffered formalin (7 days) or 4% paraformaldehyde (in 0.1 M potassium phosphate buffer, pH 7.4, 2448 hr at 4C). Postmortem heart tissue from a 42-year-old woman with systemic amyloidosis due to -light chain monoclonal gammopathy was fixed in 10% formalin for 5 days and embedded in paraffin. Paraffin blocks were prepared by sequential dehydration in graded alcohol and vacuum infiltrated in paraffin before embedding and serial sectioning to a thickness of 8 µm. Serial sections were coded and numbered to enable sequential X-34/immunohistochemical analysis. In each series, all odd-numbered sections were stained with X-34. Even-numbered sections were stained by comparison methods. In this way, each comparison stain was both preceded and followed by an X-34-stained section. Before staining, paraffin sections were taken through two 10-min washes in xylene, two 5-min washes in 100% ethanol, two 5-min washes in 95% ethanol/H2O, running tapwater for 10-min, and then 5 minutes in PBS (137 mM NaCl, 10 mM potassium phosphate, pH 7.6).
Paraformaldehyde-fixed tissue was cryoprotected by sequential incubation in 15% and 30% sucrose (in 0.1 M phosphate buffer), frozen, and cryosectioned on a Reichert Cryocut 1600 (ReichertJung; Leica, Buffalo, NY) to a thickness of 50 µm. Frozen sections were collected in Falcon 6-well plates (Fisher Scientific; Pittsburgh, PA) containing 0.1 M phosphate buffer, in a 1:6 series to allow serial analysis of X-34 and immunohistochemical and cytochemical markers. Sections were stored at 4C until assayed. Before staining, the sections were washed for 5 min in PBS.
When pretreated with formic acid, sections were soaked in 99% formic acid for 5 min and then rinsed for another 10 min in tapwater before going into the first PBS wash.
Autofluorescence Quenching Procedure and Thioflavin-S Staining
Thioflavin-S staining was performed by a modified thioflavin-S technique previously reported by
Briefly, paraffin or frozen sections to be quenched are taken from the 5-min wash in PBS and placed in a potassium permanganate solution (0.25 g% in PBS) for 20 min, after which a brown color develops. The sections are washed twice in PBS for 2 min and then treated with a solution of 1.0 g% potassium metabisulfite and 1.0 g% oxalic acid in PBS until the brown color has been removed from the tissue (typically 16 min). Finally, the sections are washed three times in PBS for 2 min each.
Bielschowsky Staining
The Bielschowsky staining method was performed according to the
X-34 General Staining Procedure
Quenched or unquenched tissue sections are taken from PBS into a solution of X-34 in 40% ethanol/60% distilled H2O (adjusted to pH 10 by addition of 1 N NaOH) at the appropriate concentration (100 nM to 1 mM) for the appropriate period of time (0.510 min). The sections are then dipped briefly five times in tapwater before differentiation in 0.2 g% NaOH in 80% ethanol for 2 min. The sections are then placed in tapwater for 10 min before coverslipping with Fluoromount-G (Electron Microscopy Science's Fort Washington, PA).
Determination of Optimal X-34 Staining
Serial 8-µm paraffin sections of frontal cortex were selected from two autopsy-confirmed AD brains. A dilution vs time matrix was constructed in which sections were stained with 100 nM, 1 µM, 10 µM, 100 µM, or 1 mM solutions of X-34. At each concentration, sections were stained for either 0.5, 1, 5, or 10 min. An identical matrix was created for quenched and unquenched sections (see above). Blind analysis was performed by three investigators (SDS, WEK, and GCS) and the optimal concentration selected. The optimal condition was defined by clear identification of resident pathology, overall image quality, and qualitative analysis of signal-to-noise ratio. Although a matrix of this type will yield a number of equivalently stained sections, preference was given to the conditions that resulted in optimal staining with the lowest concentration of X-34.
X-34 Staining/Immunohistochemistry
Alternate paraffin and cryosections were quenched as described above and stained for 10 min in 100 µM X-34 and either coverslipped or further processed for immunohistochemistry or histochemistry (
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Fluorescence Microscopy
Fluorescent sections were viewed using an Olympus Vanox AH-RFL-LB fluorescence microscope equipped with the following filter sets: U-filter (excites 360370 nm, dichroic mirror DM400, 420 nm-longpass filter); V-filter (excites 400410 nm, dichroic mirror DM455, 455-nm longpass filter); B-filter/FITC (excites 470490 nm, dichroic mirror DM500, 515-nm longpass filter); G-filter/TRITC (excites 530550 nm, dichroic mirror DM570, 590-nm longpass filter).
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Results |
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Physical Properties of X-34
X-34 (Fig 1) is a bright yellow-green solid (decomposes at 300C before melting) which is soluble at low millimolar concentrations in aqueous solution at basic or neutral pH. It is very soluble in polar organic solvents, such as DMSO. When stored as a solid (routinely stored in the dark), we have seen no evidence of deterioration in samples over 12 months old. HPLC showed a single major peak at 6.9 min (see Materials and Methods). The 500-MHz 1H NMR spectrum of X-34 in d6-DMSO (TMS reference) showed (see Fig 1 for proton numbering) H2 at 7.99 (d, J = 2.3 Hz, 2H); H6 at
7.83 (dd, J = 2.3 and 8.7 Hz, 2H); HX at
7.59 (s, 4H); HA at
7.27 (d, J = 16.3 Hz, 2H); HB at
7.14 (d, J = 16.3 Hz, 2H); H5 at
7.00 (d, J = 8.7 Hz, 2H). Mass spectrometry showed an EI parent ion of 402 as expected. The fluorescence spectra showed a broad excitation maximum at 367 nm and a broad emission maximum at 497 nm (aqueous solution at pH 7.0).
Optimization of the staining method
X-34 staining was localized to structures with the morphological appearance of Aß plaques, neurofibrillary tangles (NFTs), neuropil threads, and vascular amyloid deposits (Fig 2). X-34 staining was effective in both paraffin (Fig 2A) and frozen sections (Fig 2B). Note that, in the thick 50-µm section (Fig 2B), X-34 stains homogeneously through the entire thickness of the section, whereas antibodies often poorly penetrate sections of tissue this thick. This is best demonstrated by the homogeneous staining of short segments of two amyloid-laden blood vessels in the upper right and middle left of Fig 2B (red arrows).
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Optimal X-34 staining was observed using a violet filter cube (V-filter set). Under these conditions, X-34 fluorescence appeared as bright green/white fluorescence which was very resistant to fading with extended excitation. Different pathological entities fluoresced with slightly different colors, with NFTs and neuropil threads typically appearing cyan-white and plaques tending to appear more yellow-green (Fig 2A and Fig 2G). In many cases, this allowed easy identification of dystrophic neurites within plaques with the single stain (Fig 2A, upper inset). Vascular amyloid typically stained with such high intensity as to appear white, and often saturated the photos before other structures could be properly exposed. Analysis of fluorescence with the UV filter cube (U-filter set) revealed diminished fluorescence compared to the violet filter cube, but X-34 staining was still clearly demonstrated with this filter set. Staining was very weak using the FITC filter cube (B-filter set). X-34 did not fluoresce at all with TRITC. This profile was very useful in distinguishing specific X-34 fluorescence from autofluorescence, which was apparent with all filter sets. Therefore, the choice of filter set appears critical for optimal demonstration of X-34 fluorescence.
Analysis of the staining concentration vs staining time matrix revealed an optimal X-34 concentration of 100 µM after incubation for 10 min in quenched sections. This concentration/time combination was determined by blind analysis by three of the authors (SDS, WEK, and GCS). Sections were examined at all times and concentrations from both the quenched and unquenched matrices. In general, there were relatively small differences between the 0.5- and 10-min staining times except at the 100 nM concentration. Greater differences were observed across concentrations at a given time point. Our observations revealed that quenched sections were universally superior to unquenched sections owing to the interference of X-34 signal by lipofuscin autofluorescence. However, approximately 10-fold higher concentrations of X-34 were required to give the same intensity of staining in the quenched sections as in the unquenched sections. Although quenching produced optimal staining in our opinion, it was not essential. Using the violet filter set, unquenched lipofuscin autofluorescence was different in color (more yellowish) and morphology and could be easily distinguished from X-34 staining. In addition, X-34-specific fluorescence could be verified in unquenched sections by simply switching to the FITC or TRITC filter set in which X-34-specific fluorescence was either greatly diminished (FITC) or eliminated (TRITC), whereas the lipofuscin autofluorescence changed color but not intensity.
Although a number of concentration/time combinations revealed nearly equivalent images with high signal-to-noise ratios, the combination of 100 µM X-34 for 10 min was selected for the quenched sections. Unless otherwise noted, all reported analyses of X-34 staining of AD pathology were performed at this concentration/time combination. A combination of 10 µM X-34 for 10 min appeared optimal for unquenched sections. Because of the intensely fluorescent nature of X-34, one major factor in determining the optimal staining conditions was the amount of background fluorescence. Increased background fluorescence was observed with increasing X-34 concentration and was at least partly due to leeching of X-34 into the mounting medium. This could be best appreciated when air bubbles were present in the medium because there was virtually no background fluorescence where an air bubble overlaid the tissue.
X-34 staining by this method appears to be very stable. We have now had the opportunity to repeatedly observe the original stained sections over a period of more than 3 years. We see no apparent signs of degradation over this period of time in these X-34-stained sections (which are routinely stored in the dark).
Qualitative Comparison to Traditional Staining Methods
The apparent staining of the classical hallmarks of AD neuropathology, along with the intensity of X-34 staining and the simplicity of the method, suggested that X-34 could be useful as a sensitive stain for histochemical localization of AD pathology. Therefore, we qualitatively compared the staining of X-34 to several traditional methods of demonstrating AD neuropathology. Detailed quantitative comparisons were beyond the scope of this initial report.
Comparison of the well-established fluorescent marker of AD neuropathology, thioflavin-S, and X-34 showed that X-34 identified the same plaques, NFTs, and cerebrovascular amyloid demonstrated by thioflavin-S. In a qualitative sense, X-34 appeared to consistently stain these pathological structures more intensely than thioflavin-S. In addition, X-34 appeared to reveal plaques and NFTs not identified by thioflavin-S (Fig 3A and Fig 3C). Qualitatively, X-34 appeared to more clearly demonstrate neuropil thread pathology (insets to Fig 3A and Fig 3C). The staining by thioflavin-S was optimized by using the improved thioflavine S method for staining NFTs and senile plaques reported by
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X-34 staining also was compared to other traditional, well-characterized immunohistochemical and silver impregnation methods of identifying pathology in AD brain. It has been reported that the Bielschowsky method and Aß immunostaining are the most sensitive methods for detection of neuritic plaques (
Requirement for ß-pleated Sheets
It has been shown that staining with Congo red carries an absolute requirement for the existence of the ß-pleated sheet secondary protein structure (
X-34 Staining of Systemic Amyloidosis
Although staining with Congo red carries an absolute requirement for the existence of the ß-pleated sheet secondary protein structure, Congo red stains a variety of ß-pleated sheet amyloid deposits composed of proteins with very different primary amino acid sequences (-light chain monoclonal gammopathy (Fig 4). Compared to Congo red staining (Fig 4B), X-34 more intensely stained
-light chain amyloid (Fig 4A) and showed an almost identical pattern of distribution.
Comparison of X-34 Staining in AD and Age-matched Normal Control Brain
Whereas X-34 staining of AD brain showed an abundance of plaques, NFTs, neuropil threads, and cerebrovascular amyloid (Fig 5D5F), very little staining was observed in age-matched control brain (Fig 5A5C). Rare cerebrovascular amyloid was noted in one control brain (Fig 5B). In all control brains studied, we consistently observed small stellate profiles that appeared to be intracellular (see arrows in Fig 5A and Fig 5C). These profiles were concentrated in the molecular layer and in deep layers of neocortex, with a near absence in between. These profiles were not observed with Bielschowsky or Aß immunostaining. The morphology of these profiles was clearly distinct from that of NFTs (see arrowheads in Fig 5F), neuropil threads, and plaques. The same type of stellate profile was present in similar areas of AD brain (arrows in Fig 5E), but was almost obscured by the much more obvious network of neuropil threads, NFTs, and plaques. The very extensive neuropil staining seen in AD sections (Fig 5D5F) was absent in control brain (Fig 5A5C).
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Discussion |
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X-34 is a highly fluorescent, lipophilic derivative of Congo red that intensely stains the neuropathological hallmarks of AD brain. The staining procedure is rapid, simple, and highly reproducible. It is applicable to both fresh/frozen and formalin-fixed, paraffin-embedded sections and can be used with immunohistochemical procedures for double labeling. The use of a violet filter cube was found to be important for optimal appreciation of the high fluorescence intensity. The specific profile of X-34 fluorescence suggests that it will also be useful in co-localization studies using laser confocal microscopy. The procedure requires little technical expertise to perform and is not subject to the inherent variability associated with immunohistochemistry, thioflavin-S, or Bielschowsky silver staining. This simplicity may be one reason for the fact that our staining results have been very reproducible among several technicians in two separate laboratories. Further evaluation of the reproducibility will await the application of this method in a much larger number of laboratories.
The very low background staining indicates that a large array of ß-sheet structure is required for binding, because X-34 does not appear to stain native proteins that have only a few, relatively isolated ß-strands. Previously proposed models hypothesize that a minimum of five adjacent ß-sheet strands is necessary for Congo red (
The general affinity of X-34 for ß-sheet fibrillar deposits can be viewed as disadvantageous because it decreases the specificity of X-34 for any one particular type of deposit. However, other neuropathological characteristics, such as morphology of the deposits, topology, and associated neuropathological features, will readily distinguish among most fibrillar deposits (e.g., plaques, NFTs, and cerebrovascular amyloid). The general affinity for ß-sheet deposits also can be viewed as an advantage in the study of AD brain. In some studies, it may be technically advantageous to intensely stain both plaque and tangle pathology with a single simple method. This is particularly true if other double-labeling procedures are to be performed. In many circumstances, it would be advantageous to simultaneously identify and perhaps even quantitate a more comprehensive measure of the AD pathology than either plaques alone or NFT alone. X-34 allows the simultaneous demonstration of plaques (neuritic and non-neuritic), cerebrovascular amyloid, NFTs, and neuropil threads. In most AD cases, the extents of all of these neuropathological changes are highly correlated with one another other and the cumulative assessment may give a better measure of the effective progression of the disease than would any one of the pathological entities examined in isolation.
This intense staining of AD pathology by X-34 raises the question of whether X-34 staining might identify pathological structures in other neurodegenerative or neuropsychiatric diseases. For example, it would be expected that X-34 would stain plaques in prion disease, which have been shown to contain ß-sheet fibrillar peptides (-light chain monoclonal gammopathy supports the notion that X-34 is a general amyloid stain. Along with the inhibition of X-34 staining after disruption of the ß-pleated sheet fibril secondary structure with formic acid, this indicates that, like Congo red, X-34 is more specific for ß-pleated sheet secondary structure than for the primary amino acid sequence of the amyloid protein.
Our results from X-34-stained elderly control brains (Fig 5A5C) have shown very little X-34 staining, supporting the specificity of X-34 for pathological fibrillization of endogenous proteins. This, coupled with the lipophilic nature of X-34, supports further pursuit of our original goal of developing X-34 or its derivatives as in vivo neuroimaging agents (using PET or SPECT for detection of radiolabeled derivatives of X-34) in living AD patients (
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Footnotes |
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1 Current address: Aventis Pharmaceuticals, Bridgewater, NJ.
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Acknowledgments |
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Supported by the Alzheimer's Association (IIRG-95-076, WEK) and Aventis Pharmaceuticals. RLH was supported in part by the University of Pittsburgh Alzheimer's Disease Research Center (NIA-AG-05133).
We wish to thank Manik L. Debnath for his work on the synthetic chemistry components, and Yetta Wilbur and Jonette Werley for their contribution to the histopathology studies. Portions of this work were performed in facilities made available by Drs Jay Pettegrew, Steven DeKosky, and Clayton Wiley at the University of Pittsburgh.
Received for publication October 11, 1999; accepted April 5, 2000.
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