Abstract
Immuno-electron microscopy and electron microscopic in situ hybridization are powerful tools to identify the precise subcellular localization of specific proteins and RNAs at the ultramicroscopic level. Here we describe detailed procedures for how to detect the precise location of a specific target labeled with both fluorescence and gold particles. Although they have been developed for the analysis of Drosophila ovarian somatic cells, these techniques are suitable for a wide range of biological applications including human, primate, and rodent analysis.
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References
Siomi MC, Sato K, Pezic D et al (2011) PIWI-interacting small RNAs: the vanguard of genome defence. Nat Rev Mol Cell Biol 12:246–258
Juliano C, Wang J, Lin H (2011) Uniting germline and stem cells: the function of Piwi proteins and the piRNA pathway in diverse organisms. Annu Rev Genet 45:447–469
Ishizu H, Siomi H, Siomi MC (2012) Biology of PIWI-interacting RNAs: new insights into biogenesis and function inside and outside of germlines. Genes Dev 26:2361–2373
Malone CD, Brennecke J, Dus M et al (2009) Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary. Cell 137:522–535
Brennecke J, Aravin AA, Stark A et al (2007) Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128:1089–1103
Vagin VV, Sigova A, Li C et al (2006) A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313:320–324
Saito K, Nishida KM, Mori T et al (2006) Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev 20:2214–2222
Aravin A, Gaidatzis D, Pfeffer S et al (2006) A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442:203–207
Gunawardane LS, Saito K, Nishida KM et al (2007) A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila. Science 315:1587–1590
Saito K, Ishizu H, Komai M et al (2010) Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila. Genes Dev 24:2493–2498
Olivieri D, Sykora MM, Sachidanandam R et al (2010) An in vivo RNAi assay identifies major genetic and cellular requirements for primary piRNA biogenesis in Drosophila. EMBO J 29:3301–3317
Handler D, Olivieri D, Novatchkova M et al (2011) A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors. EMBO J 30:3977–3993
Olivieri D, Senti KA, Subramanian S et al (2012) The cochaperone shutdown defines a group of biogenesis factors essential for all piRNA populations in Drosophila. Mol Cell 47:954–969
Qi H, Watanabe T, Ku HY et al (2011) The Yb body, a major site for Piwi-associated RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells. J Biol Chem 286:3789–3797
Szakmary A, Reedy M, Qi H et al (2009) The Yb protein defines a novel organelle and regulates male germline stem cell self-renewal in Drosophila melanogaster. J Cell Biol 185:613–627
Murota Y, Ishizu H, Nakagawa S et al (2014) Yb integrates piRNA intermediates and processing factors into perinuclear bodies to enhance piRISC assembly. Cell Rep 8:103–113
Matsuno A, Nagashima T, Ohsugi Y et al (2000) Electron microscopic observation of intracellular expression of mRNA and its protein product: technical review on ultrastructural in situ hybridization and its combination with immunohistochemistry. Histol Histopathol 15:261–268
Herrera GA (1992) Ultrastructural immunolabeling: a general overview of techniques and applications. Ultrastruct Pathol 16:37–45
Zhang L, Kaneko S, Kikuchi K et al (2014) Rewiring of regenerated axons by combining treadmill training with semaphorin3A inhibition. Mol Brain 7:14
Takano M, Kawabata S, Komaki Y et al (2014) Inflammatory cascades mediate synapse elimination in spinal cord compression. J Neuroinflammation 11:40
Numasawa-Kuroiwa Y, Okada Y, Shibata S et al (2014) Involvement of ER stress in dysmyelination of Pelizaeus-Merzbacher disease with PLP1 missense mutations shown by iPSC-derived oligodendrocytes. Stem Cell Rep 2:648–661
Nishimoto Y, Nakagawa S, Hirose T et al (2013) The long non-coding RNA nuclear-enriched abundant transcript 1_2 induces paraspeckle formation in the motor neuron during the early phase of amyotrophic lateral sclerosis. Mol Brain 6:31
Takano M, Hikishima K, Fujiyoshi K et al (2012) MRI characterization of paranodal junction failure and related spinal cord changes in mice. PLoS One 7, e52904
Yasuda A, Tsuji O, Shibata S et al (2011) Significance of remyelination by neural stem/progenitor cells transplanted into the injured spinal cord. Stem Cells 29:1983–1994
Nagoshi N, Shibata S, Hamanoue M et al (2011) Schwann cell plasticity after spinal cord injury shown by neural crest lineage tracing. Glia 59:771–784
Tada H, Okano HJ, Takagi H et al (2010) Fbxo45, a novel ubiquitin ligase, regulates synaptic activity. J Biol Chem 285:3840–3849
Kumagai G, Okada Y, Yamane J et al (2009) Roles of ES cell-derived gliogenic neural stem/progenitor cells in functional recovery after spinal cord injury. PLoS One 4, e7706
Saito K (2014) RNAi and overexpression of genes in ovarian somatic cells. Methods Mol Biol 1093:25–33
Acknowledgments
We are grateful to Dr. S. Nakagawa for providing insightful discussions and dedicated support to our project. We thank T. Yano at Electron microscope laboratory and G. Itai at Keio-med Open Access Facility for their special technical support and also thank all members of the Siomi and Okano laboratories for their invaluable comments. This work was supported by a Grant-in-Aid for Scientific Research from MEXT, Japan; a grant from Keio Gijuku Academic Development Funds to S.S.; and a grant from Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) to S.S. and H.O. The authors have no conflicts of interest to declare.
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Shibata, S. et al. (2015). Immuno-Electron Microscopy and Electron Microscopic In Situ Hybridization for Visualizing piRNA Biogenesis Bodies in Drosophila Ovaries. In: Bratu, D., McNeil, G. (eds) Drosophila Oogenesis. Methods in Molecular Biology, vol 1328. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2851-4_12
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DOI: https://doi.org/10.1007/978-1-4939-2851-4_12
Publisher Name: Humana Press, New York, NY
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