title: Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification.
abstract: 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 alpha2 (GFRalpha2) 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 GFRalpha2 mRNA and virtually all GFRalpha2-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 GFRalpha2 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.
authors.affiliation: Divisions of Neuropathology, Department of Pathology, Washington University School of Medicine, St Louis, Missouri, USA.
authors.forename: A U
authors.initials: AU
authors.lastname: Zaidi
authors.forename: H
authors.initials: H
authors.lastname: Enomoto
authors.forename: J
authors.initials: J
authors.lastname: Milbrandt
authors.forename: K A
authors.initials: KA
authors.lastname: Roth
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000033:anatomy & histology / Q000254:growth & development / Q000378:metabolism; D029721:Drosophila Proteins; D005629:Frozen Sections; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D007150:Immunohistochemistry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D016612:Paraffin Embedding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret; D012333:RNA, Messenger / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D012668:Trigeminal Ganglion / Q000378:metabolism; D014439:Tyramine
chemical_list: D029721:Drosophila Proteins; C494991:Gfra2 protein, mouse; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; C494999:Ret protein, mouse; D014439:Tyramine