Protocols for two- and three-color fluorescent RNA in situ hybridization of the main and accessory olfactory epithelia in mouse

Abstract

The main and accessory olfactory epithelia of the mouse are composed of many cell populations. Each sensory neuron is thought to express one allele of one of the approximately 1000 odorant or approximately 300 vomeronasal receptor genes. Sensory neurons die and are replaced by new neurons that differentiate from precursor cells throughout the lifetime of the individual. Neuronal replacement is asynchronous, resulting in the co-existence of cells at various stages of differentiation. Receptor gene diversity and ongoing neuronal differentiation produce complex mosaics of gene expression within these epithelia. Accurate description of gene expression patterns will facilitate the understanding of mechanisms of gene choice and differentiation. Here we report a detailed protocol for two- and three-color fluorescent RNA in situ hybridization (ISH) and its combination with immunohistochemistry, or detection of bromodeoxyuridine (BrdU)-incorporated DNA after labeling. The protocol is applied to cryosections of the main and accessory olfactory epithelia in mouse.