Using Combined Fluorescent In Situ Hybridization With Immunohistochemistry to Co-localize mRNA in Diverse Neuronal...

Understanding gene expression within defined neuronal populations is essential for dissecting the cellular and molecular diversity of the brain. mRNA assays provide a direct readout of gene expression, capturing transcriptional changes that may precede or occur independently of protein abundance, whereas protein assays reflect the cumulative effects of translation, modification, and degradation. Moreover, in histological analysis, immunohistochemical protein detection results in visually diffuse labeling, which makes it difficult to quantitatively assess levels and locations of expression at high resolution. Here, we present a protocol that allows for mRNA detection in single neuronal cell types with a high degree of sensitivity and anatomical resolution. This protocol combines fluorescent in situ hybridization (FISH) with immunohistochemistry (IHC) on the same tissue section. Briefly, FISH is carried out by ACDBio RNAscope^® fluorescent in situ hybridization technology, which involves processing the tissue sections, followed by signal amplification. This involves target retrieval, probe hybridization, and signal enhancement. Then, the tissue section is processed for IHC, which involves blocking nonspecific sites and incubation with primary antibodies, followed by development of a fluorescent signal with secondary antibodies. Typically, visual mRNA detection with FISH can be seen as individual puncta, whereas targeting the protein with an antibody results in filled cells or processes. The variation in staining pattern allows for the quantification of distinct mRNA transcripts within different neuronal populations, which renders co-localization analyses easy and efficient. Key features • This protocol combines ACDBio RNAscope^TM technology with standard immunohistochemistry techniques. • Visual simultaneous detection of mRNA and protein expression within murine brain tissue to identify mRNA transcripts within specific neuronal subpopulations. • Using a custom macro code for FIJI/ImageJ for an efficient analysis of total mRNA expression or mRNA co-localization with neuronal subtypes. • This protocol can be used to detect any mRNA and protein combination given appropriate antigen retrieval and tissue preparation within any tissue type of interest.