title: Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization.
abstract: mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.
authors.affiliation: College of Physicians and Surgeons, Columbia University.
authors.forename: Jimena
authors.initials: J
authors.lastname: Baleriola
authors.affiliation: College of Physicians and Surgeons, Columbia University.
authors.forename: Ying
authors.initials: Y
authors.lastname: Jean
authors.affiliation: College of Physicians and Surgeons, Columbia University.
authors.forename: Carol
authors.initials: C
authors.lastname: Troy
authors.affiliation: College of Physicians and Surgeons, Columbia University; uh2112@cumc.columbia.edu.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Hengst
mesh_terms: D051701:Activating Transcription Factor 4 / Q000235:genetics; D000818:Animals; D001369:Axons / Q000737:chemistry*; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis*
chemical_list: C497109:ATF4 protein, human; C081452:Atf4 protein, mouse; D012333:RNA, Messenger; D051701:Activating Transcription Factor 4