Protease-Free RNAscope and Sequential Immunofluorescence Methods for Manual Codetection of RNAs and Proteins

In situ hybridization (ISH) is an important method used in the visualization of gene expression/regulation signatures in cell and tissues. The ability to codetect mRNA and protein within the same tissue section on a single slide by combining proteomic immunohistochemistry (IHC) and transcriptomic ISH technologies has allowed for detailed spatial multiomic mapping within individual cells and their microenvironment. In this chapter, we describe the manual application of a universal permeabilization protease-free reagent, PretreatPro™, with RNAscope™ amplification and detection of mRNA in FFPE tissue samples for codetection of mRNA and protein without loss of antigenicity. PretreatPro is compatible with both chromogenic and fluorescent detection modalities when amplified with RNAscope.