Abstract
We describe here a high-sensitivity in situ hybridization protocol, optimized for fresh-frozen brain sections, that enables the detection of two transcripts, at single cell resolution. Riboprobes directed against two mRNAs of interest are synthesized with nucleotides tagged with different haptens (digoxigenin- or biotin-UTP), via in vitro transcription, hybridized simultaneously to brain sections, and independently detected through immunocytochemistry. Sequential detection of each probe involves peroxidase-mediated precipitation of tyramide-linked fluorophores of separate emission wavelengths. In addition, we demonstrate how classic non-fluorescent chromogens, such as 3,3′-diaminobenzidine, can be successfully combined with fluorescence-based detection, to yield reliable detection of two transcript populations. We provide examples of representative results obtained with this protocol and describe necessary controls. Additionally, we discuss common problems associated with this methodology, and detail troubleshooting recommendations. Although this method has been optimized for brain sections, it may be useful to detect two mRNA species in a variety of tissues.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pinaud, R. and Tremere, L.A. (2006) Immediate early genes in sensory processing, cognitive performance and neurological disorders, Pinaud, R. and Tremere, L.A. (eds.), Springer-Verlag, New York.
Pinaud, R., Tremere, L.A., and De Weerd, P. (2005) Plasticity in the visual system: from genes to circuits, Pinaud, R., Tremere, L.A., and De Weerd, P. (eds.), Springer-Verlag, New York.
Pinaud, R. (2004) Experience-dependent immediate early gene expression in the adult central nervous system: evidence from enriched-environment studies. Int. J. Neurosci. 114, 321–333.
Ooi, L. and Wood, I.C. (2008) Regulation of gene expression in the nervous system. Biochem. J. 414, 327–341.
Navarro, X., Vivo, M., and Valero-Cabre, A. (2007) Neural plasticity after peripheral nerve injury and regeneration. Prog. Neurobiol. 82, 163–201.
Bading, H. (1999) Nuclear calcium-activated gene expression: possible roles in neuronal plasticity and epileptogenesis. Epilepsy Res. 36, 225–231.
Thomas, G.M. and Huganir, R.L. (2004) MAPK cascade signalling and synaptic plasticity. Nat. Rev. Neurosci. 5, 173–183.
Cohen, S. and Greenberg, M.E. (2008) Communication between the synapse and the nucleus in neuronal development, plasticity, and disease. Annu. Rev. Cell Dev. Biol. 24, 183–209.
Greene, J.G. (2006) Gene expression profiles of brain dopamine neurons and relevance to neuropsychiatric disease. J. Physiol. 575, 411–416.
Jin, L. and Lloyd, R.V. (1997) In situ hybridization: methods and applications. J. Clin. Lab. Anal. 11, 2–9.
Stoler, M.H. (1990) In situ hybridization. Clin. Lab. Med. 10, 215–236.
Komminoth, P. and Werner, M. (1997) Target and signal amplification: approaches to increase the sensitivity of in situ hybridization. Histochem. Cell Biol. 108, 325–333.
Pinaud, R., Velho, T.A., Jeong, J.K., Tremere, L.A., Leão, R.M., von Gersdorff, H., and Mello, C.V. (2004) GABAergic neurons participate in the brain’s response to birdsong auditory stimulation. Eur. J. Neurosci. 20, 1318–1330.
Kessler, C. (1991) The digoxigenin:anti-digoxigenin (DIG) technology – a survey on the concept and realization of a novel bioanalytical indicator system. Mol. Cell. Probes 5, 161–205.
Panoskaltsis-Mortari, A. and Bucy, R.P. (1995) In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts. Biotechniques 18, 300–307.
Qian, X. and Lloyd, R.V. (2003) Recent developments in signal amplification methods for in situ hybridization. Diagn. Mol. Pathol. 12, 1–13.
Velho, T.A., Pinaud, R., Rodrigues, P.V., and Mello, C.V. (2005) Co-induction of activity-dependent genes in songbirds. Eur. J. Neurosci. 22, 1667–1678.
Pinaud, R., Mello, C.V., Velho, T.A., Wynne, R.D., and Tremere, L.A. (2008) Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization. Nat. Protoc. 3, 1370–1379.
Mello, C.V., Jarvis, E.D., Denisenko, N., and Rivas, M. (1997) Isolation of song-regulated genes in the brain of songbirds. Methods Mol. Biol. 85, 205–217.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Pinaud, R., Jeong, J.K. (2010). Duplex In Situ Hybridization in the Study of Gene Co-regulation in the Vertebrate Brain. In: Hewitson, T., Darby, I. (eds) Histology Protocols. Methods in Molecular Biology, vol 611. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-345-9_9
Download citation
DOI: https://doi.org/10.1007/978-1-60327-345-9_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-344-2
Online ISBN: 978-1-60327-345-9
eBook Packages: Springer Protocols