title: Dual enhancement of double immunofluorescent signals by CARD: participation of ubiquitin during formation of neurofibrillary tangles.
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-Abeta 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 Abeta 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.
authors.affiliation: Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Japan. uchihara@tmin.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Uchihara
authors.forename: A
authors.initials: A
authors.lastname: Nakamura
authors.forename: U
authors.initials: U
authors.lastname: Nagaoka
authors.forename: M
authors.initials: M
authors.lastname: Yamazaki
authors.forename: O
authors.initials: O
authors.lastname: Mori
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism* / Q000473:pathology*; D016229:Amyloid beta-Peptides / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D000918:Antibody Specificity; D001710:Biotin / Q000031:analogs & derivatives; D001921:Brain / Q000378:metabolism / Q000473:pathology; D002384:Catalysis; D000939:Epitopes / Q000032:analysis; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D016874:Neurofibrillary Tangles / Q000378:metabolism* / Q000473:pathology*; D014439:Tyramine / Q000031:analogs & derivatives; D014452:Ubiquitins / Q000378:metabolism*; D016875:tau Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D000939:Epitopes; D005456:Fluorescent Dyes; D014452:Ubiquitins; C111551:biotinyltyramide; D016875:tau Proteins; D001710:Biotin; D016650:Fluorescein-5-isothiocyanate; D014439:Tyramine