Skip to main content
SpringerLink
Log in

Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

  • Protocol
  • Published:

From Nature Protocols

View current issue Submit your manuscript

Abstract

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2–3 d.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Double-fluorescence in situ hybridization (dFISH) flowchart.
Figure 2: Representative double-fluorescence in situ hybridization (dFISH) results.
Figure 3: Key equipment and setup necessary to conduct dFISH.

Similar content being viewed by others

References

  1. Pinaud, R. Critical calcium-regulated biochemical and gene expression programs involved in experience-dependent plasticity. in Plasticity in the Visual System: From Genes to Circuits (eds. Pinaud, R., Tremere, L.A. & De Weerd, P.) 153–180 (Springer-Verlag, New York, 2005).

    Google Scholar 

  2. Pinaud, R. & Tremere, L.A. Immediate Early Genes in Sensory Processing, Cognitive Performance and Neurological Disorders (Springer-Verlag, New York, 2006).

    Book  Google Scholar 

  3. Pinaud, R., Tremere, L.A. & De Weerd, P. Plasticity in the Visual System: From Genes to Circuits (Springer-Verlag, New York, 2005).

    Google Scholar 

  4. Hatten, M.E. & Heintz, N. Large-scale genomic approaches to brain development and circuitry. Annu. Rev. Neurosci. 28, 89–108 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Gustincich, S. et al. The complexity of the mammalian transcriptome. J. Physiol. 575, 321–332 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hofmann, H.A. Functional genomics of neural and behavioral plasticity. J. Neurobiol. 54, 272–282 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Pascual-Leone, A., Amedi, A., Fregni, F. & Merabet, L.B. The plastic human brain cortex. Annu. Rev. Neurosci. 28, 377–401 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Hensch, T.K. Critical period regulation. Annu. Rev. Neurosci. 27, 549–579 (2004).

    Article  CAS  Google Scholar 

  9. Buonomano, D.V. & Merzenich, M.M. Cortical plasticity: from synapses to maps. Annu. Rev. Neurosci. 21, 149–186 (1998).

    Article  CAS  Google Scholar 

  10. Yao, H. & Dan, Y. Synaptic learning rules, cortical circuits, and visual function. Neuroscientist 11, 206–216 (2005).

    Article  PubMed  Google Scholar 

  11. Chklovskii, D.B., Mel, B.W. & Svoboda, K. Cortical rewiring and information storage. Nature 431, 782–788 (2004).

    Article  CAS  Google Scholar 

  12. Sanes, J.N. & Donoghue, J.P. Plasticity and primary motor cortex. Annu. Rev. Neurosci. 23, 393–415 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Weinberger, N.M. Dynamic regulation of receptive fields and maps in the adult sensory cortex. Annu. Rev. Neurosci. 18, 129–158 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Singh, S.M., Treadwell, J., Kleiber, M.L., Harrison, M. & Uddin, R.K. Analysis of behavior using genetical genomics in mice as a model: from alcohol preferences to gene expression differences. Genome 50, 877–897 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Chambers, D. & Fishell, G. Functional genomics of early cortex patterning. Genome Biol. 7, 202 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Clayton, D.F. Songbird genomics: methods, mechanisms, opportunities, and pitfalls. Ann. NY Acad. Sci. 1016, 45–60 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Tribl, F. et al. Proteomics of the human brain: sub-proteomes might hold the key to handle brain complexity. J. Neural. Transm. 113, 1041–1054 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Pinaud, R., Osorio, C., Alzate, O. & Jarvis, E.D. Profiling of experience-regulated proteins in the songbird auditory forebrain using quantitative proteomics. Eur. J. Neurosci. 27, 1409–1422 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Kee, N., Teixeira, C.M., Wang, A.H. & Frankland, P.W. Imaging activation of adult-generated granule cells in spatial memory. Nat. Protoc. 2, 3033–3044 (2007).

    Article  CAS  PubMed  Google Scholar 

  20. Guzowski, J.F. et al. Mapping behaviorally relevant neural circuits with immediate-early gene expression. Curr. Opin. Neurobiol. 15, 599–606 (2005).

    Article  CAS  Google Scholar 

  21. Terleph, T.A. & Tremere, L.A. The use of immediate early genes as mapping tools for neuronal activation: concepts and methods. in Immediate Early Genes in Sensory Processing, Cognitive Performance and Neurological Disorders (eds. Pinaud, R. & Tremere, L.A.) 1–10 (Springer-Verlag, New York, 2006).

    Google Scholar 

  22. Guzowski, J.F. Immediate early genes and the mapping of environmental representations in hippocampal networks. in Immediate Early Genes in Sensory Processing, Cognitive Performance and Neurological Disorders (eds. Pinaud, R. & Tremere, L.A.) 159–176 (Springer-Verlag, New York, 2006).

    Chapter  Google Scholar 

  23. Owens, N.C., Hess, F.M. & Badoer, E. In situ hybridization using riboprobes on free-floating brain sections. Methods Mol. Biol. 326, 163–171 (2006).

    CAS  PubMed  Google Scholar 

  24. Henderson, Z. In Situ Hybridization Techniques for the Brain (John Wiley & Sons, New York, 1996).

    Google Scholar 

  25. Schneider Gasser, E.M. et al. Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons. Nat. Protoc. 1, 1887–1897 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Glynn, M.W. & McAllister, A.K. Immunocytochemistry and quantification of protein colocalization in cultured neurons. Nat. Protoc. 1, 1287–1296 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Wisden, W. & Morris, B.J. In Situ Hybridization Protocols for the Brain (Academic Press Inc., San Diego, 1994).

    Google Scholar 

  28. Zaidi, A.U., Enomoto, H., Milbrandt, J. & Roth, K.A. Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification. J. Histochem. Cytochem. 48, 1369–1375 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Guzowski, J.F., McNaughton, B.L., Barnes, C.A. & Worley, P.F. Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles. Nat. Neurosci. 2, 1120–1124 (1999).

    Article  CAS  Google Scholar 

  30. Breininger, J.F. & Baskin, D.G. Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification. J. Histochem. Cytochem. 48, 1593–1599 (2000).

    Article  CAS  PubMed  Google Scholar 

  31. Velho, T.A., Pinaud, R., Rodrigues, P.V. & Mello, C.V. Co-induction of activity-dependent genes in songbirds. Eur. J. Neurosci. 22, 1667–1678 (2005).

    Article  PubMed  Google Scholar 

  32. Pinaud, R. et al. GABAergic neurons participate in the brain's response to birdsong auditory stimulation. Eur. J. Neurosci. 20, 1318–1330 (2004).

    Article  PubMed  Google Scholar 

  33. Pinaud, R., Penner, M.R., Robertson, H.A. & Currie, R.W. Upregulation of the immediate early gene arc in the brains of rats exposed to environmental enrichment: implications for molecular plasticity. Brain Res. Mol. Brain Res. 91, 50–56 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Pinaud, R. et al. Complexity of sensory environment drives the expression of candidate-plasticity gene, nerve growth factor induced-A. Neuroscience 112, 573–582 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Pinaud, R., Saldanha, C.J., Wynne, R.D., Lovell, P.V. & Mello, C.V. The excitatory thalamo-'cortical' projection within the song control system of zebra finches is formed by calbindin-expressing neurons. J. Comp. Neurol. 504, 601–618 (2007).

    Article  PubMed  Google Scholar 

  36. Mello, C.V., Jarvis, E.D., Denisenko, N. & Rivas, M. Isolation of song-regulated genes in the brain of songbirds. Methods Mol. Biol. 85, 205–217 (1997).

    CAS  PubMed  Google Scholar 

  37. Mello, C.V. & Clayton, D.F. Differential induction of the ZENK gene in the avian forebrain and song control circuit after metrazole-induced depolarization. J. Neurobiol. 26, 145–161 (1995).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The work described here was supported by NIH/NIDCD and a start-up package from the University of Rochester to R.P. R.P. would like to dedicate this work to the memory of Manoel Campos Ribeiro.

Author information

Authors and Affiliations

  1. Department of Brain and Cognitive Sciences, University of Rochester, Rochester, 14627, New York, USA

    Raphael Pinaud, Ryan D Wynne & Liisa A Tremere

  2. Center for Visual Science, University of Rochester, Rochester, 14627, New York, USA

    Raphael Pinaud

  3. Neurological Sciences Institute, Oregon Health and Science University, Portland, 97006, Oregon, USA

    Claudio V Mello & Tarciso A Velho

Authors
  1. Raphael Pinaud
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Claudio V Mello
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tarciso A Velho
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ryan D Wynne
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Liisa A Tremere
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Raphael Pinaud.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pinaud, R., Mello, C., Velho, T. et al. Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization. Nat Protoc 3, 1370–1379 (2008). https://doi.org/10.1038/nprot.2008.115

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2008.115

  • Springer Nature Limited

This article is cited by

Search

Navigation