Abstract
The use of tagged RNA probes to directly hybridize frozen sections of chemically fixed tissues, followed by the tag detection with specific antibodies and gold conjugates form the core of the in situ hybridization (ISH)-immunoelectron microscopy (IEM) method that we have developed and successfully used to detect endogenous gurken and bicoid mRNAs in Drosophila oocytes.
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References
Lawrence JB, Singer RH (1986) Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell 45(3):407–415
Grunert S, St Johnston D (1996) RNA localization and the development of asymmetry during Drosophila oogenesis. Curr Opin Genet Dev 6(4):395–402
Riechmann V, Ephrussi A (2001) Axis formation during Drosophila oogenesis. Curr Opin Genet Dev 11(4):374–383
Slot JW, Geuze HJ (2007) Cryosectioning and immunolabeling. Nat Protoc 2(10):2480–2491. doi:10.1038/nprot.2007.365
Herpers B, Xanthakis D, Rabouille C (2010) ISH-IEM: a sensitive method to detect endogenous mRNAs at the ultrastructural level. Nat Protoc 5(4):678–687. doi:10.1038/nprot.2010.12
Delanoue R, Herpers B, Soetaert J, Davis I, Rabouille C (2007) Drosophila Squid/hnRNP helps Dynein switch from a gurken mRNA transport motor to an ultrastructural static anchor in sponge bodies. Dev Cell 13(4):523–538. doi:10.1016/j.devcel.2007.08.022
Weil TT, Parton RM, Herpers B, Soetaert J, Veenendaal T, Xanthakis D, Dobbie IM, Halstead JM, Hayashi R, Rabouille C, Davis I (2012) Drosophila patterning is established by differential association of mRNAs with P bodies. Nat Cell Biol 14(12):1305–1313. doi:10.1038/ncb2627
Herpers B, Rabouille C (2004) mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in gurken transport in Drosophila oocytes. Mol Biol Cell 15(12):5306–5317. doi:10.1091/mbc.E04-05-0398
Howley PM, Israel MA, Law MF, Martin MA (1979) A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes. J Biol Chem 254(11):4876–4883
Le Guellec D (1998) Ultrastructural in situ hybridization: a review of technical aspects. Biol Cell 90(4):297–306
Acknowledgments
I thank Dr. Bram Herpers (Leiden, NL) in collaboration with Ilan Davis and his group (Oxford, UK), and Tim Weil (Cambridge, UK) for developing and using this technique.
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Rabouille, C. (2018). Detection of mRNA and Associated Molecules by ISH-IEM on Frozen Sections. In: Gaspar, I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7213-5_11
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DOI: https://doi.org/10.1007/978-1-4939-7213-5_11
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7212-8
Online ISBN: 978-1-4939-7213-5
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