MMISH: Multicolor microRNA in situ hybridization for paraffin embedded samples

Abstract To understand and assess the roles of miRNAs, visualization of the expression patterns of specific miRNAs is needed at the cellular level in a wide variety of different tissue types. Although miRNA in situ hybridization techniques have been greatly improved in recent years, they remain difficult to routinely perform due to the complexity of the procedure. In addition, as it is crucial to define which tissues or cells are expressing a particular miRNA in order to elucidate the biological function of the miRNA, incorporation of additional stainings for different cellular markers is necessary. Here, we describe a robust and flexible multicolor miRNA in situ hybridization (MMISH) technique for paraffin embedded sections. We show that the miRNA in situ protocol is sensitive and highly specific and can successfully be combined with both immunohistochemical and immunofluorescent stainings.