59 Charting the murine immune landscape: high-plex spatial profiling across diverse tissues
Year: 2026
Type: conference-abstract
Abstract: Background The immune system is a highly dynamic network whose function depends on the precise spatial organization and interaction of heterogeneous cell populations within native tissue architectures. 1 Traditional Methods like flow cytometry capture cellular abundance but lose critical spatial context. While multiplex immunofluorescence provides a solution, characterizing the complex murine immune landscape across multiple distinct organs has historically been hindered by long optimization timelines, tissue degradation, and rigid antibody requirements. This study aimed to address these constraints by developing a high-plex panel of murine immune markers to use in combination with automated staining technology.Methods A comprehensive 16-marker panel targeting key T cell subsets, B cells, macrophages, dendritic cells, and structural tissue markers was developed, optimized, and run across diverse FFPE mouse tissues (i.e., spleen, lymph nodes, lung, gut). Biomarkers included in the panel were as follows: MEF2A, F4/80, aSMA, CD31, CD45, Tim-3, CD4, CD3, CD20, FoxP3, EGFR, Lag-3, COL1A1, COL4A1, PD-1, and CD8. Utilizing the Lunaphore COMET™ integrated staining and imaging platform allowed us to leverage off-the-shelf, un-conjugated primary antibodies. Following the automated run, samples were counterstained with hematoxylin and eosin for tissue architecture visualization.Results Validation of this 16-marker panel on the COMET™ platform demonstrated highly robust, reproducible, and fully automated high-plex staining across all tested mouse tissues. By utilizing off-the-shelf primary antibodies, we bypassed traditional secondary antibody species crosstalk, enabling uniform signal amplification for all markers. Signal-to-noise ratios remained consistently high, with no observable signal dimming across sequential staining cycles. Additionally, the automated run completed the entire 16-marker sequence for all tissue samples in under 36 hours, significantly reducing hands-on optimization time compared to manual cyclic immunofluorescence protocols. Concordant staining patterns were achieved for low-abundance immune checkpoints (PD-1, Lag-3) and high-density structural targets (COL4A1, aSMA), confirming that sequential elution cycles did not compromise epitope availability. Finally, the counterstaining of samples revealed no artifact introduction and preserved structural architecture ( figure 1).Conclusions Our findings demonstrate that this 16-plex assay in combination with the COMET™ platform provides a rapid, robust, and highly flexible workflow for broad spatial profiling of the murine immune system. The automation of the instrument, high-plex nature of the assay, and gentle staining procedure allow for additional downstream tissue use. Additionally, the lack of antibody barcoding or conjugation significantly accelerated assay development. This workflow could serve as a valuable pipeline for evaluating tissue-specific immune responses in models of infection, inflammation, and cancer immunotherapy.Reference Navarro QR, Villarreal CJ, Zarate PE, et al. Multiscale information processing in the immune system. Front Immunol. 2025;16:1563992.Abstract 59 Figure 1Mouse spleen tissue (A) is stained with MEF2A (red), F4/80 (green), CD31 (white), aSMA (yellow), and DAPI (blue). A region of interest (B) is chosen to display the high density, but distinct populations, of positive F4/80 and MEF2A cells
Authors: Danielle M Fails, Alyssa Hernandez
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,490
OpenAlex ID: W7225335121