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ISH–IEM: a sensitive method to detect endogenous mRNAs at the ultrastructural level

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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Figure 1: Flow chart and timing of in situ hybridization (ISH)–immuno-electron microscopy (IEM) on semi-thin (indicated in red) and ultrathin frozen sections.
Figure 2: In situ hybridization (ISH)–immuno-electron microscopy (IEM) on ultrathin sections is dependent on hybridization buffer composition.
Figure 3: gurken mRNA localization in stage 9 Drosophila oocyte by in situ hybridization (ISH)–immuno-electron microscopy (IEM).
Figure 4: oskar mRNA localization in stage 10 Drosophila oocyte by in situ hybridization (ISH)–immuno-electron microscopy (IEM).

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Acknowledgements

We thank our colleagues of the Department of Cell Biology (UMC Utrecht), Jan Willem Slot, Jan van Minnen, Ilan Davis, Renald Delanoue and Anne Ephrussi for helpful discussions and reagents. C.R. wishes to thank her doctors from the Diakonessenziekenhuis Utrecht for putting her back in good health. B.H. was funded by a Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) Aspasia grant (015.001.129) to C.R.

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B.H. developed the protocol. D.X. refined some of the steps and C.R. supervised the progress and provided expertise.

Corresponding author

Correspondence to Catherine Rabouille.

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Herpers, B., Xanthakis, D. & Rabouille, C. ISH–IEM: a sensitive method to detect endogenous mRNAs at the ultrastructural level. Nat Protoc 5, 678–687 (2010). https://doi.org/10.1038/nprot.2010.12

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