ArticleNature protocols2010
ISH-IEM: a sensitive method to detect endogenous mRNAs at the ultrastructural level.
Article in Nature protocols, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed, 25 citations in OpenAlex.
- Patterning the Drosophila embryo: A paradigm for RNA-based developmental genetic regulation.Wiley interdisciplinary reviews. RNA · 2020Review
- The viral replication organelles within cells studied by electron microscopy.Advances in virus research · 2019Article
- Localized Translation of gurken/TGF-α mRNA during Axis Specification Is Controlled by Access to Orb/CPEB on Processing Bodies.Cell reports · 2016Article
- Drosophila germ granules are structured and contain homotypic mRNA clusters.Nature communications · 2015Article
- Independent and coordinate trafficking of single Drosophila germ plasm mRNAs.Nature cell biology · 2015Article
- Methods for studying oogenesis.Methods (San Diego, Calif.) · 2014Review
- Article
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Authors and funding
3 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
This protocol describes the combination of in situ hybridization (ISH) with cryo-immunolabeling methods to allow the simultaneous detection at the ultrastructural level of mRNAs and proteins. The procedure consists of five steps and takes 4-5 d: (i) acquisition of ultrathin frozen sections of chemically fixed tissues or cells; (ii) hybridization of the sections with digoxigenin (DIG) or biotin-labeled RNA probes; (iii) detection of the bound probe with antibodies and protein A-gold (PAG); (iv) labeling of proteins of interest (optional); and (v) visualization by transmission electron microscopy (immuno-electron microscopy (IEM)). This technique allows the simultaneous detection of endogenous/overexpressed/injected RNAs and proteins while preserving the cell ultrastructure. The protocol is also suitable for mRNA detection on semi-thin frozen sections in combination with immunofluorescence. The localization of targeted transcripts, such as gurken and oskar mRNA in the Drosophila oocyte, and of structural elements and proteins that mediate their localization have been revealed using this technique.
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Registered trials
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