Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification

To understand the biological relationships among various molecules, it is n ecessary to define the cellular expression patterns of multiple genes and g ene products. Relatively simple methods for performing multi-label immunohi stochemical detection are available. However, there is a paucity of techniq ues for dual immunohistochemical (IHC) and mRNA in situ hybridization (ISH) detection. The recent development of improved non-radioactive detection sy stems and simplified ISH protocols has prompted us to develop a tyramide si gnal amplification method for sequential multi-label fluorescent ISH and IH C detection in either frozen or paraffin-embedded tissue sections. We used this method to examine the relationship between glial cell line-derived neu rotrophic factor receptor alpha 2 (GFR alpha 2) mRNA expression and IHC loc alization of its co-receptor Ret in the trigeminal ganglion of postnatal Da y 0 mice. We found that approximately 70% of Ret-immunoreactive neurons pos sessed GFR alpha 2 mRNA and virtually all GFR alpha 2-expressing neurons co ntained Ret-immunoreactive protein. Finally, we used paraformaldehyde-fixed , paraffin-embedded sections and a monoclonal antibody against neuron-speci fic nuclear antigen (NeuN) to demonstrate the neuronal specificity of GFR a lpha 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 l ocalization.