Precise Visualization of Insulin Receptors A and B in Murine Brain with an RNA In Situ Hybridization Assay

Insulin receptors (IRs) are widely expressed across all tissues and all cell types. It exists in two functionally distinct isoforms -- IR-A and IR-B -- differing by 12 amino acids due to alternative exon 11 splicing. These isoforms exhibit tissue- and cell-specific expression patterns, with their relative abundance varying throughout development, aging, and disease. Due to their high sequence similarity, distinguishing between IR-A and IR-B using traditional immunological methods has been challenging. Here, we describe a novel Duplex in situ hybridization (ISH) assay that enables the visualization of IR-A and IR-B expression in tissue. Using this approach, we demonstrate for the first time that while both isoforms are expressed in the murine choroid plexus, IR-A is predominant. This method provides a powerful tool for investigating the spatial distribution of insulin receptor isoforms in situ across different tissues. While real-time qPCR can detect IR-A and IR-B transcripts in monocellular in vitro cultures, it lacks the capacity for spatial localization within complex tissues, highlighting the need for and utility of the Duplex ISH technique.