Abstract
The localization of specific mRNAs into dendrites and/or axons is an important mechanism to enrich proteins at their sites of function and influence neuronal development, plasticity, and repair. The fluorescence in situ hybridization (FISH) methods described here have provided high sensitivity and resolution enabling investigation into the mechanism, regulation, and function of mRNA localization in vitro and in vivo. Two methods are described in detail. The first method employs digoxigenin- or fluorophore-conjugated oligonucleotide probes for the detection of localized mRNAs in dendrites, spines, axons, and growth cones of cultured neurons. The second method employs digoxigenin-labeled RNA probes and fluorescence tyramide amplification for the detection of less abundant mRNAs localized to dendrites in vivo. Both methods enable the visualization and quantification of mRNA granules, and changes in their localization in response to various stimuli. The high-resolution FISH technology described here has broader applications beyond the study of mRNA localization. It enables the quantitative analyses of developmental and cell type-specific patterns of gene expression, and how these are modified by physiological signals or during disease states.
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Acknowledgment
The authors thank several past and current members of the Bassell lab for their efforts to refine and optimize this FISH technology. This work was supported by MH085617 and HD055835 to GJB, a postdoctoral fellowship and Conquer Fragile X research grant from the National Fragile X Foundation to C.G., and predoctoral fellowships F31NS063668, T32GM0860512 and T32NS007480, and the Epilepsy Foundation and Lennox & Lombroso Trust Fund to S.A.S.
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Swanger, S.A., Bassell, G.J., Gross, C. (2011). High-Resolution Fluorescence In Situ Hybridization to Detect mRNAs in Neuronal Compartments In Vitro and In Vivo. In: Gerst, J. (eds) RNA Detection and Visualization. Methods in Molecular Biology, vol 714. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-005-8_7
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DOI: https://doi.org/10.1007/978-1-61779-005-8_7
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