Dual enhancement of double immunofluorescent signals by CARD: participation of ubiquitin during formation of neurofibrillary tangles.

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.