Background Spatial biology methods are increasingly used for the characterization of complex tissue microenvironments, the understanding of which can shed light on fundamental biological mechanisms and better inform development of targeted therapeutics.

RNAscope™ in situ hybridization (ISH) technology, capable of highly sensitive single-molecule RNA detection, can be combined with immunohistochemistry (IHC) or immunofluorescence (IF) for the co-detection of clinically relevant biomarkers on the same slide with morphological context. This application is especially important in immuno-oncology research to profile immune cell populations using protein markers and characterize their activation states by detecting cytokine and chemokine expression with RNA. However, RNAscope necessitates the use of proteases to digest RNA-associated proteins and facilitate probe access to RNA targets, which can negatively impact epitopes targeted by some antibodies.

Previously, we developed the Integrated Co-detection Workflow (ICW) to partially solve this problem, with the fixation of the primary antibody-target complex prior to the protease application. While ICW rescues signal for many previously incompatible antibodies, proteases can still adversely impact some of the primary antibody-target complex. To address this deficiency, we have developed a novel RNA-protein co-detection workflow that eliminates the need for protease, resulting in high detection sensitivities for both protein and RNA markers.

Methods To maintain the same RNA detection sensitivity without the use of proteases, we formulated a new protease-free pretreatment buffer to replace the existing protease step within the current workflow which allows adequate accessibility of RNAscope™ probes to the target RNAs. Following this protease-free pretreatment, tissue specimens were assayed with RNAscope Multiplex to detect RNA species in the tissue, followed by standard IF staining to co-detect protein biomarkers. Antibodies which previously exhibited degraded protein signal in both sequential ISH-IF and ICW were tested in the new protocol.

Results Here, we present results from FFPE human tissues to co-detect several protease-sensitive antibodies, including degranulating cytotoxic lymphocyte marker CD107a along with human house-keeping genes TBP, POLR2A and PPIB. The protease-free RNAscope co-detection workflow restored the protein staining pattern at nominal antibody concentrations used for IHC while maintaining mRNA dot counts for TBP, POLR2A and PPIB, indicating minimal impact of protease-free pretreatment buffer on both RNA and protein signal.

Conclusions The protease-free RNAscope co-detection workflow will serve as a powerful multi-omics staining technique for a wider range of antibodies by enabling visualization of RNA-protein co-detection for the comprehensive profiling of tissue microenvironments, facilitating faster breakthroughs in the discovery of therapeutics.