title: Regulation of Neurotrophic Factors During Pathogenic Tau-Aggregation: A Detailed Protocol for Double-Labeling mRNA by In Situ Hybridization and Protein Epitopes by Immunohistochemistry.
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.
authors.affiliation: Institute of Applied Biotechnology, University of Applied Sciences Biberach, Karlstrasse 11, 88400, Biberach/Riss, Germany. zimmermann@hochschule-bc.de.
authors.forename: Katharina
authors.initials: K
authors.lastname: Schindowski-Zimmermann
mesh_terms: D003704:Dementia / Q000378:metabolism; D000939:Epitopes / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D009414:Nerve Growth Factors / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000737:chemistry*; D024801:Tauopathies / Q000378:metabolism*; D016875:tau Proteins / Q000032:analysis / Q000378:metabolism*
chemical_list: D000939:Epitopes; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D016875:tau Proteins
keywords: Dementia; Double staining; Immunofluorescence; Immunohistochemistry; In situ hybridization; Neurotrophins; Riboprobe synthesis; Tau hyperphosphorylation; Tauopathy