Abstract
The conclusive demonstration of RNA in vertebrate axons by in situ hybridization (ISH) has been elusive. We review the most important reasons for difficulties, including low concentration of axonal RNAs, localization in specific cortical domains, and the need to isolate axons. We demonstrate the importance of axon micro-dissection to obtain a whole mount perspective of mRNA distribution in the axonal territory. We describe a protocol to perform fluorescent ISH in isolated axons and guidelines for the preservation of structural and molecular integrity of cortical RNA-containing domains (e.g., Periaxoplasmic Ribosomal Plaques, or PARPs) in isolated axoplasm.
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Acknowledgments
The authors thank John Mercer for a critical reading of this manuscript and Edward Koenig for the use of figures presented in Fig. 1. This project was partially funded by CSIC, PEDECIBA, ANII, MEyC, PEW fellowship to JRSS, NIH grant #1R03 TW007220-01A2.
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Sotelo-Silveira, J.R., Calliari, A., Kun, A., Elizondo, V., Canclini, L., Sotelo, J.R. (2011). Localization of mRNA in Vertebrate Axonal Compartments by In Situ Hybridization. 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_8
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DOI: https://doi.org/10.1007/978-1-61779-005-8_8
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