Molecular Instruments

HCR™ Pro RNA-ISH enables high-resolution visualization of RNA expression within tissue sections, offering a powerful tool for studying immune cell dynamics. Here, PD-L1, a key immune checkpoint ligand, is explored as both an RNA (imaged in red) and protein (imaged in green) marker to investigate its expression patterns in normal human tonsil tissue.

 With its diverse immune cells and organized lymphoid structure, the normal tonsil is an ideal model for studying immune regulation. Detecting PD-L1 at both the RNA and protein levels within the same sample reveals the interplay between transcriptional activity and protein expression, shedding light on this critical marker’s role in immune homeostasis. 

This integrated RNA and IHC approach maximizes the use of valuable samples, enabling a comprehensive analysis of PD-L1’s spatial expression and functional dynamics. Such insights are essential for advancing our understanding of immune checkpoint biology in disease, paving the way for the development of more effective, targeted immunotherapies.

One of the key advantages of HCR™ Pro RNA-ISH is its protease-free nature, which preserves tissue morphology while maintaining high specificity for RNA detection. This makes it ideally suited for seamless integration with IHC and IF assays, allowing researchers to co-detect RNA and protein markers within the same tissue section, without compromising structural integrity. 

In this example, the use of an HCR™ Membrane Stain (imaged in green) highlights the precise segmentation of cellular membranes, enabling clear and distinct visualization of Ppib RNA (imaged in red) expression patterns along the cell boundaries. By combining HCR™ Pro RNA-ISH with IHC/IF assays, researchers can gain a deeper understanding of cellular interactions and gene expression patterns.

This AACR collaboration with Leica Biosystems, Molecular Instruments, Abcam, and Indica Labs highlights an end-to-end workflow for profiling the colorectal tumor microenvironment in FFPE tissue. On the BOND RX research staining system, Abcam antibody clones were first evaluated in single-plex format using HCR™ Pro IHC and shown to produce staining patterns comparable to BOND Polymer Refine Detection on serial sections. Building on that validation, HCR™ Gold IF enabled same-species multiplex immunofluorescence without iterative staining, supporting flexible panel development while preserving tissue morphology.

Using HALO^® for tissue segmentation, cell phenotyping, density measurements, and spatial analysis, the workflow distinguished healthy colon from colorectal cancer based on epithelial organization, immune cell distribution, and proliferative activity. Multiplex analysis of panCK, Ki67, CD3, and CD68 showed enrichment of tumor and proliferating tumor cells in CRC epithelium, with immune populations more abundant in stromal regions and altered T-cell localization at the tumor- stroma interface. Together, the poster shows how automated HCR™ workflows plus quantitative image analysis can support scalable, spatially resolved tumor microenvironment studies.

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