Automated ISH for Validated Histological Mapping of Lowly Expressed Genes

An essential part of the drug discovery and development process in the pharmaceutical industry is to provide a full characterization of cells expressing a given drug target and potential downstream markers in human tissues and in relevant preclinical animal species. This task is best solved by a combination of methods, including histological assessment of target protein and mRNA using immunohistochemistry (IHC) and in situ hybridization (ISH), respectively, as well as non-histology-based methods, such as fluorescence-activated cell sorting (FACS), and single-cell (SCS) or single-nuclei (SNS) sequencing. In reality, this work is often complicated by a combination of low target expression levels and a less than optimal availability of specific reagents for detection. In particular, the ability to specifically detect low-abundance receptor targets using IHC is notoriously difficult, due to a daunting lack of commercially available specific antibodies validated for use in IHC. In the absence of fully validated antibodies and protocols for IHC, the specific detection of target mRNA using ISH is often the only available histological method. A highly sensitive, nonradioactive, automated, and robust ISH method for use on formalin-fixed, paraffin-embedded (FFPE) tissue sections is presented for assessing histological localization of mRNA transcripts of lowly expressed genes.