Tau Protein pp 391-414 | Cite as

Regulation of Neurotrophic Factors During Pathogenic Tau-Aggregation: A Detailed Protocol for Double-Labeling mRNA by In Situ Hybridization and Protein Epitopes by Immunohistochemistry

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1523)

Abstract

Alzheimer’s disease (AD), most tauopathies, and other neurodegenerative diseases are highly associated with impaired neurotrophin regulation and imbalanced neutrophin distribution. Tau phosphorylation occurs at different sites of the tau protein and some phospho-epitopes are associated with normal ageing (like tau phosphorylated at Ser202/Thr205 detected by the antibody clone AT8) and others are highly associated with AD (abnormally phosphorylated tau at Thr212/Ser214 detected by the antibody clone AT100). Neurotrophins are crucial for the survival and maintenance of distinct neuronal population; therefore, their supply is essential for a healthy brain. Though their importance is well known, their analysis in tissue is not trivial and needs careful consideration. Here, a detailed a protocol is presented, how to combine in situ hybridization (ISH) with immunohistochemistry (IHC) to analyze neurotrophins during tau neuropathology and the results were confirmed by immunological methods. In addition, the preparation of the riboprobes is presented step-by-step. Since there are growing evidences for the relevance of neurotrophic factor distribution in the pathogenesis of AD, this technique is one useful tool to investigate the underlying mechanisms and therapeutic intervention.

Key words

Double staining In situ hybridization Immunohistochemistry Immunofluorescence Riboprobe synthesis Neurotrophins Tauopathy Dementia Tau hyperphosphorylation 
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Notes

Acknowledgments

This study was performed at Sanofi-Aventis, Vitry-sur-Seine/France, supported from a Marie Curie Fellowship from the European Community in collaboration with Dr. Thomas Rooney, Sanofi Sarl, Vitry-sur-Seine/France and Dr. Luc Buée, Inserm, Lille/France. A special thanks goes to Uwe Ernsberger for transfer of the ISH technique and to Klaus Unsicker for transfer of skills on neuroanatomy and on the importance of neuroanatomy for neuroscience.

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Institute of Applied BiotechnologyUniversity of Applied Sciences BiberachBiberach/RissGermany

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