caa a22 4500 475774078 CHVBK 20180406123625.0 cr unu---uuuuu 170329e20001201xx s 000 0 eng 10.1007/s004180000218 doi (NATIONALLICENCE)springer-10.1007/s004180000218 Dual enhancement of double immunofluorescent signals by CARD: participation of ubiquitin during formation of neurofibrillary tangles [Elektronische Daten] [Toshiki Uchihara, Ayako Nakamura, Utako Nagaoka, Mineo Yamazaki, Osamu Mori] Abstract.: Amplification with catalyzed reporter deposition (CARD) greatly enhances peroxidase signals, which has been utilized to amplify immunohistochemical labelings including fluorochromes. Here we describe a strategy to amplify each of two immunofluorescent signals without crosstalk on double-stained histological sections from human autopsied brains with Alzheimer's disease (AD). One of the two primary antibodies (anti-Aβ or anti-PHF-tau) was probed by a species-specific secondary antibody conjugated with horseradish peroxidase (HRP), which was visualized by FITC-labeled tyramide. After inactivation of HRP, the other primary antibody was probed by another species-specific secondary antibody conjugated with HRP. Amplification with biotinylated tyramide was followed by streptavidin-conjugated Cy-5, which specifically labeled the latter epitope. It was found that Aβ and PHF-tau were localized to senile plaques and neurofibrillary tangles (NFTs), respectively, which verified lack of crosstalk on the double-stained section. Localization of ubiquitin and PHF-tau was looked for at higher magnification in NFT-bearing neurons. Although these two epitopes were colocalized in some neurons, ubiquitin was not always present in PHF-tau positive NFTs. Discrepancy between PFH-tau and ubiquitin, verified inter- and intracellularly, may represent different stages of NFT formation. This is the first report of successful CARD amplification of two different fluorescent signals on double-labeling immunohistochemistry, which is now proved to be powerful in detecting epitopes in relation to AD-related lesions. Improved intensity over tenfold of the two fluorescent signals without crosstalk will expand the application of the multilabeling method with fluorochromes. Springer-Verlag, 2000 Multifluorescence CARD Immunohistochemistry Tyramide Confocal nationallicence Uchihara Toshiki Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashi-dai, Fuchu, Tokyo, 183-8526, Japan aut Nakamura Ayako Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashi-dai, Fuchu, Tokyo, 183-8526, Japan aut Nagaoka Utako Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashi-dai, Fuchu, Tokyo, 183-8526, Japan aut Yamazaki Mineo Second Department of Internal Medicine, Nippon Medical School, 1-5-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan aut Mori Osamu Second Department of Pathology, Nippon Medical School, 1-5-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan aut https://doi.org/10.1007/s004180000218 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s004180000218 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Uchihara Toshiki Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashi-dai, Fuchu, Tokyo, 183-8526, Japan aut NATIONALLICENCE 700 1- Nakamura Ayako Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashi-dai, Fuchu, Tokyo, 183-8526, Japan aut NATIONALLICENCE 700 1- Nagaoka Utako Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashi-dai, Fuchu, Tokyo, 183-8526, Japan aut NATIONALLICENCE 700 1- Yamazaki Mineo Second Department of Internal Medicine, Nippon Medical School, 1-5-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan aut NATIONALLICENCE 700 1- Mori Osamu Second Department of Pathology, Nippon Medical School, 1-5-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan aut Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer