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Journal of Histochemistry and Cytochemistry, Vol. 51, 1201-1206, September 2003, Copyright © 2003, The Histochemical Society, Inc.

ARTICLE

Triple Immunofluorolabeling with Two Rabbit Polyclonal Antibodies and a Mouse Monoclonal Antibody Allowing Three-dimensional Analysis of Cotton Wool Plaques in Alzheimer Disease

Toshiki Uchiharaa, Ayako Nakamuraa, Hiroshi Nakayamac, Kunimasa Arimad, Norio Ishizukab, Hiroshi Morie, and Setsuo Mizushimaf
a Departments of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan
b Brain Structure, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan
c Department of Psychiatry, Tokyo Metropolitan Neurological Hospital, Tokyo, Japan
d Department of Laboratory Medicine, National Center Hospital for Mental, Nervous, and Muscular Disorders, National Center for Neurology and Psychiatry, Tokyo, Japan
e Department of Neuroscience, Osaka City University School of Medicine, Osaka, Japan
f Mizushima Clinic, Tokyo, Japan

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

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 I–III, and was sometimes in contact with the vascular wall, both without neuritic reactions. CWPs, present in layers I–VI, 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 51:1201–1206, 2003)

Key Words: triple immunofluorescence, three dimensions, reconstruction, laser confocal microscopy, amyloid, Aß42, cotton wool plaques


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