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Journal of Histochemistry and Cytochemistry, Vol. 48, 1369-1376, October 2000, Copyright © 2000, The Histochemical Society, Inc.

ARTICLE

Dual Fluorescent In Situ Hybridization and Immunohistochemical Detection with Tyramide Signal Amplification

Aliya U. Zaidia, Hideki Enomotob, Jeffrey Milbrandtb, and Kevin A. Rotha
a Divisions of Neuropathology, Department of Pathology, Washington University School of Medicine, St Louis, Missouri
b Laboratory Medicine, Department of Pathology, Washington University School of Medicine, St Louis, Missouri

Correspondence to: Kevin A. Roth, Washington U. School of Medicine, Dept. of Pathology, Div. of Neuropathology, 660 S. Euclid Avenue, Box 8118, St Louis, MO 63110. E-mail: kroth@pathology.wustl.edu

To understand the biological relationships among various molecules, it is necessary to define the cellular expression patterns of multiple genes and gene products. Relatively simple methods for performing multi-label immunohistochemical detection are available. However, there is a paucity of techniques for dual immunohistochemical (IHC) and mRNA in situ hybridization (ISH) detection. The recent development of improved non-radioactive detection systems and simplified ISH protocols has prompted us to develop a tyramide signal amplification method for sequential multi-label fluorescent ISH and IHC detection in either frozen or paraffin-embedded tissue sections. We used this method to examine the relationship between glial cell line-derived neurotrophic factor receptor {alpha}2 (GFR{alpha}2) mRNA expression and IHC localization of its co-receptor Ret in the trigeminal ganglion of postnatal Day 0 mice. We found that approximately 70% of Ret-immunoreactive neurons possessed GFR{alpha}2 mRNA and virtually all GFR{alpha}2-expressing neurons contained Ret-immunoreactive protein. Finally, we used paraformaldehyde-fixed, paraffin-embedded sections and a monoclonal antibody against neuron-specific nuclear antigen (NeuN) to demonstrate the neuronal specificity of GFR{alpha}2 mRNA expression in adult mouse brain. This multi-labeling technique should be applicable to a wide variety of tissues, antibodies, and probes, providing a relatively rapid and simple means to compare mRNA and protein localization. (J Histochem Cytochem 48:1369–1375, 2000)

Key Words: fluorescent in situ hybridization, tyramide signal amplification, immunohistochemistry, double labeling


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