9-channel spatial co-detection of clinically relevant protein and RNA targets in FFPE tumors with HCR™ Gold and MoxiePlex

Background: High-plex spatial profiling of protein & RNA in FFPE tumors is increasingly important for characterizing tumor-immune interactions, but existing methods often limit exploration to predefined antibody panels, harsh stripping/eluting steps, and iterative cycles that compromise sample and target integrity and make assay development and validation burdensome. HCR™ Gold IF, enabled by the HiFi Encoder, supports enzyme-free fluorescent detection using unmodified, user-supplied primary antibodies and operates on the same amplification platform as HCR™ Gold RNA-FISH. Combined with the Hamamatsu MoxiePlex multispectral tissue imaging platform, which enables whole-slide single-round imaging of 7-9-plex panels without the need for cyclic-staining, this workflow offers a practical route to flexible, 7-9-plex protein and RNA co-detection in FFPE specimens. Methods: FFPE human tumor sections were stained with HCR™ Gold IF using encoded, clinically validated primary antibodies alongside HCR™ HiFi Probes. Two parallel multiplex configurations were tested on the MoxiePlex:1. 7-plex panel: DAPI, 425, 488, 546, 594, 700, 7502. 9-plex panel: DAPI, 425, 488, 514, 546, 594, 633, 700, 750Panels incorporated antibodies specific to cancer-relevant proteins (e.g., pan-CK, Ki-67, PD-L1, CD8, CD68) & RNA targets representing tumor and immune biology (e.g., EPCAM, CXCL9, CD274). For each fluorophore-target combination, single-stain reference slides were generated to establish exposure settings, verify filter performance, and quantify spectral bleed-through to enable spectral unmxing. Multiplex slides were imaged in a single acquisition round, and data were evaluated for signal intensity, channel crosstalk, expected biological localization, and concordance with single-stain references. Spectral unmixing was carried out automatically for the whole- slide image, following acquisition, as part of the imaging process. Results: HCR™ Gold IF & HCR™ Gold RNA-FISH produced bright, robust signals with preserved tissue architecture. In whole- slide multiplex imaging, protein and RNA targets localized to expected tumor and stroma regions highlighting immune infliltration in the tumor microenvironment, the expression of which. closely matched single-stain reference patterns. The MoxiePlex platform delivered consistent, whole-slide imaging performance with reproducible acquisition settings appropriate for routine 7-9-plex scanning. Conclusions: HCR™ Gold IF & HCR™ Gold RNA-FISH can be combined in a single-round workflow on the MoxiePlex to achieve up to 9-plex imaging of clinically relevant protein and RNA targets in FFPE tissue. This approach avoids stripping or photobleaching, maintains compatibility with validated antibodies and standard filter sets, and provides a practical path for flexible, 7-9-plex spatial assays in translational and clinical research.