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Fluorescent mRNA labeling through cytoplasmic FISH

Nature Protocols volume 8, pages 2538–2547 (2013)Cite this article

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Abstract

RNA in situ hybridization (ISH) has been widely used in cell and developmental biology research to study gene expression. Classical ISH protocols use colorimetric staining approaches, such as the assay with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP), which do not allow the implementation of multiple probe analyses and do not enable investigators to achieve cellular resolution. Here we describe a protocol to determine the presence of target cytoplasmic RNA via cytoplasmic fluorescence ISH (cFISH), an approach that renders possible the visualization of specific RNA strands from the whole tissue down to the cell. This fluorescence technique, adapted here for use in mouse embryos, enables researchers to implement multiple labeling by combining several RNA probes and/or antibodies in immuno-cFISH. Depending on the options chosen, the protocol can be completed within 2 or 3 d.

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Figure 1: Workflow of the protocol.
Figure 2: Schematic representation of cFISH and amplified cFISH experiments.
Figure 3: Expected results from the different procedures.
Figure 4: Expected results for combined cFISH experiments on mouse embryos of different stages.

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Acknowledgements

These protocols were originally developed by C.C. in J. Rossant's laboratory, on the basis of a chromogenic protocol13. We thank B. Ciruna for the initial use of tyramide amplification. This work has been supported by the 'Ligue Nationale contre le Cancer- CD 63' and by the Agence Nationale de la Recherche (ANR) 'EpiNodal'. C.D. was supported with a fellowship from the ANR 'EpiNodal'. The adult and fetal human tissues were obtained from the Brain and Tissue Bank for Developmental Disorders at the University of Maryland.

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Author notes

  1. Maxime Gasnier and Cynthia Dennis: These authors contributed equally to this work.

Authors and Affiliations

  1. INSERM, U1103, Laboratoire Génétique, Reproduction et Développement (GreD), Clermont-Ferrand, France

    Maxime Gasnier, Cynthia Dennis, Catherine Vaurs-Barrière & Claire Chazaud

  2. CNRS, UMR6293, Laboratoire GReD, Clermont-Ferrand, France

    Maxime Gasnier, Cynthia Dennis, Catherine Vaurs-Barrière & Claire Chazaud

  3. Clermont Université, Laboratoire GReD, Clermont-Ferrand, France

    Maxime Gasnier, Cynthia Dennis, Catherine Vaurs-Barrière & Claire Chazaud

Authors
  1. Maxime Gasnier
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  2. Cynthia Dennis
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  3. Catherine Vaurs-Barrière
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  4. Claire Chazaud
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Contributions

M.G., C.D. and C.C. wrote the manuscript; C.C. designed the protocols; M.G. and C.D. performed the experiments on mouse embryos and C.V.-B. on human brain tissues; and C.C. supervised the project.

Corresponding author

Correspondence to Claire Chazaud.

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The authors declare no competing financial interests.

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Gasnier, M., Dennis, C., Vaurs-Barrière, C. et al. Fluorescent mRNA labeling through cytoplasmic FISH. Nat Protoc 8, 2538–2547 (2013). https://doi.org/10.1038/nprot.2013.160

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