ARTICLE |
Correspondence to: Toshiki Uchihara, Dept. of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashi-dai, Fuchu, Tokyo 183-8526, Japan. E-mail: uchihara@tmin.ac.jp
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Summary |
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We established a triple-labeling method with two rabbit polyclonal antibodies and a mouse monoclonal antibody and examined autopsied brain tissue with cotton wool plaques (CWPs). One of the polyclonal antibodies was so diluted (anti-Aß42 or anti-Aß40/1:30,000 or anti-von Willebrand factor/1:1000) that its visualization was possible only after amplification with the catalyzed reporter deposition (CARD) method. The other polyclonal antibody (anti-Aß40 or anti-Aß42/1:1000) was visualized with a fluorochrome conjugated to an anti-rabbit antibody that specifically visualized the latter polyclonal antibody because of its lower sensitivity. A monoclonal antibody, AT8, was superimposed to yield triple immunofluorolabeling. Serial optical sections with an interval of 0.3 µm were reconstructed to allow three-dimensional (3D) observation of these three epitopes. Aß40 was localized to core-like structures, mainly in layers IIII, and was sometimes in contact with the vascular wall, both without neuritic reactions. CWPs, present in layers IVI, were labeled with anti-Aß42 and were accompanied by neuritic reactions. These differences suggest that mechanisms of Aß deposition and its relation to neuritic reactions or to blood vessels differ according to the lesion, even in the same microscopic field.
(J Histochem Cytochem 51:12011206, 2003)
Key Words: triple immunofluorescence, three dimensions, reconstruction, laser confocal microscopy, amyloid, Aß42, cotton wool plaques
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Introduction |
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THE COTTON WOOL PLAQUE (CWP) is a peculiar type of senile plaque seen in brains of some patients with familial Alzheimer disease (AD) and in those of sporadic AD. CWPs are characterized by robust deposition of amyloid ß protein (Aß), Aß42 (
One of the obstacles was that the antibodies to be applied on the same sections were from the same species, which usually hampers multilabeling. Several procedures have been proposed to circumvent this difficulty, with some success. For example, conjugation of enzyme, biotin, or fluorochrome to one of the primary antibodies allows separate detection of the two antibodies, but it requires a relatively large amount of the antibody and the conjugation procedure can be cumbersome (
A previous study demonstrated that double immunofluorolabeling with two antibodies from the same species was possible with extensive blockade with F(ab')2 fragment (
In observing structures, such as senile plaques, that exceed the thickness (510 µm) of routine histological sections, we are not sure whether or not portions included in the section under observation represent the entire structure. We therefore obtained serial optical sections under a laser scanning confocal microscope to be reconstructed for 3D observation. Three-dimensional reconstruction of triple-labeled sections, as we established in this study, can provide an opportunity to observe the entire structure of CWPs and the spatial relationship between the relevant structures.
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Materials and Methods |
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A dementia patient with a familial background of Alzheimer disease was diagnosed with AD based on the presence of many senile plaques and neurofibrillary tangles. Senile plaques in this case were not clearly detectable with the Bodian method, but hematoxylin and eosin staining visualized the cotton wool feature of the plaques without a core. In addition, a core-like structure and perivascular deposits of Aß were observed (
We first undertook experiments to establish the optimal concentrations of the antibodies for the methods used, especially the dilution of the primary antibodies visualized with the CARD method but undetectable by the standard method without CARD amplification. The list of primary antibodies is provided in Table 1. Three-µm-thick mirror sections were obtained from formalin-fixed, paraffin-embedded blocks from the occipital lobe of this patient. Deparaffinized sections were treated with formic acid (>99%) for 5 min to enhance Aß-like immunoreactivity (
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For triple immunofluorolabeling, deparaffinized sections were similarly subjected to immunolabeling with CARD amplification with one of the polyclonal antibodies made in rabbits (anti-Aß42 1:30,000; anti-Aß40 1:30,000; or anti-von Willebrand factor 1:1000; DAKO, Glostrup, Denmark) as described above and finally visualized to Cy5 conjugated to streptavidin (1:200; Kirkegaard & Perry, Gaithersburg, MD). von Willebrand factor is a marker for vascular endothelial cells and the anti-von Willebrand factor antibody immunolabels blood vessels (
For 3D observation, formalin-fixed blocks were washed in PBS and cryoprotected by being soaked in 20% sucrose buffered with 0.1 M phosphate. Thick floating sections (50100-µm in thickness) were obtained on a freezing microtome. They were subjected to the triple-immunolabeling method as above with prolonged incubation (up to 7 days) with the primary antibody. Sections were mounted with 90% glycerol in 0.1 M phosphate buffer containing 0.1% of p-phenylenediamine and were observed under a confocal laser scanning microscope (Leica TCS/SP; Heidelberg, Germany). Excitation of the fluorochromes and their maximal detection wavelength are summarized in Table 2. Serial optical sections were obtained and reconstructed for 3D analysis on software (TRI/3D; Ratoc System, Tokyo, Japan).
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Results |
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Serial dilution of one of the polyclonal antibodies (anti-Aß40) demonstrated that the usual immunofluorescence method without CARD amplification (Fig 1A, Fig 1C, Fig 1E, and Fig 1G) requires a high concentration of the antibody (1:1000) to obtain maximal labeling (Fig 1C). However, CARD amplification (Fig 1B, Fig 1D, Fig 1F, and Fig 1H) enabled us to use the antibody at lower concentrations (up to 1:30,000) to obtain a clear fluorescent signal of an equivalent intensity with little background staining (Fig 1F). Omission of either primary antibody, secondary antibody, or biotinylated tyramide completely eliminated the immunofluorescent signal. Similar results were obtained with the anti-Aß42 antibody (data not shown).
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Therefore, we first performed CARD-amplified immunofluorolabeling with anti-Aß40 (Fig 2A2C), which was visualized with Cy5, shown as blue. Subsequent double labeling with AT8 (1:1000; Fig 2A2C, red) and with anti-Aß42 (1:1000; Fig 2A and Fig 2C, green) antibody was visualized with the mixture of anti-mouse IgG conjugated with rhodamine and anti-rabbit IgG conjugated with FITC. As shown in Fig 2, no crossreaction between the two rabbit polyclonal antibodies (anti-Aß40 and anti-Aß42) was detectable. Moreover, it is noteworthy that reverse experiments, anti-Aß42 with amplification followed by anti-Aß40, gave essentially the same results (Fig 3).
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Most of the CWPs were homogeneously labeled with the anti-Aß42 antibody [green in Fig 2 (arrows) and blue in Fig 3], and participation of Aß40 was partial on these CWPs (blue in Fig 2 and green in Fig 3). In contrast, Aß40 was deposited as core-like structures (arrowheads in Fig 2C and Fig 3), which were rare in deeper layers of the cerebral cortices (asterisk in Fig 2B, blue) and contained also Aß42 (arrowheads in Fig 2C). Three-dimensional analysis demonstrated that these core-like structures positive for Aß40 (Fig 4, green) were sometimes clustered along the blood vessel (arrowhead in Fig 4A). The Aß40 epitope was sometimes co-localized to the vessel wall, with an occasional continuity to these core-like structures. Neuritic reactions, detected with AT8/rhodamine, were present around CWPs (asterisks in Fig 3, red), but they were rarely observed around the core-like structures (arrowheads in Fig 3) or the vessel wall (arrow in Fig 3).
As suggested by the stacked view of the 114 optical sections (Fig 4A), some of the core-like structures were occasionally clustered around blood vessels. Three-dimensional observation with the software enabled us to examine a simultaneous stereoscopic view of these three epitopes (Aß40, PHF-tau, and blood vessel). Finally, a cross-sectional view along an arbitrary cutting line on the blood vessel (from arrowhead to asterisk in Fig 4) showed that Aß40 accumulated around the blood vessel either in a linear or a spherical fashion (Fig 4E).
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Discussion |
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We successfully performed triple immunofluorolabeling with two antibodies raised in rabbits and another mouse MAb. Single immunofluorolabeling with different concentrations of the anti-Aß40 antibody clarified that the optimal dilution of the anti-Aß antibodies without CARD amplification was 1:1000, whereas equivalent labeling was obtained at a dilution of 1:30,000 after amplification with CARD. One of the theoretical bases for discriminating two distinct epitopes with antibodies raised in the same species depends on this difference in the optimal concentration of the antibody for immunofluorescence study (
These triple-labeled sections demonstrated that most of the CWPs were homogeneously stained with the anti-Aß42 antibody (
In summary, we established a triple-immunofluorolabeling method with two rabbit polyclonal antibodies and a mouse monoclonal antibody, which simultaneously visualized Aß40, Aß42, or von Willebrand factor and PHF-tau epitopes. Application of this triple immunofluorolabeling enabled thick sections to be analyzed on a 3D basis. Immunohistochemical features and distribution were found to be different between Aß42-positive deposits and their Aß40-positive counterparts. This suggests that the mechanism of Aß deposition for CWPs is different from that of other Aß deposits, such as core-like structures. This triple-labeling method will expand the applicability and precision of multiple immunoflurolabeling, which is advantageous in a wide range of research and diagnosis.
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Acknowledgments |
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Supported in part by a grant (TU) from the Ministry of Culture, Sports, Science and Technology, Japan.
We are grateful to Mr Ray Cowan for reading the manuscript.
Received for publication November 5, 2002; accepted April 2, 2003.
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