OMAP-21: Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of Palatine Tonsil (FFPE) with MICS on MACSima, v1.0

OMAP-21 was designed for MICS (MACSima imaging cyclic staining) imaging of FFPE human tonsil samples. Tissue fixation<br>and antigen retrieval is described in (Spatial protein and RNA analysis on the same tissue section using MICS<br>technology). The MACSima technology is described in detail in the following publication (MACSima imaging cyclic<br>staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors). Most, but not<br>all, antibodies in this panel are recombinant antibodies with a mutated human IgG1 constant region. The described<br>mutation removes the Fc receptor binding capacity of human IgG1, eliminating the need for additional blocking steps<br>and reducing non-specific binding of human antibodies on human tissues. Highly multiplexed imaging is achieved through<br>cycles of immunolabeling with FITC, PE, and APC conjugated antibodies and photobleaching to eliminate fluorescence<br>signal between imaging cycles. The panel contains 47 antibodies and the nuclear marker DAPI for image alignment and<br>nuclear segmentation. This OMAP provides a spatial context for all anatomical structures and most cell types present<br>in the human palatine tonsil. OMAP-21 follows closely OMAP-1 described for human lymph nodes<br>(omap-1-human-lymph-node-ibex) and OMAP-10 (omap-10-palatine-tonsil-macsima). All reagents were obtained from<br>Miltenyi Biotec and have been rigorously tested through an internal quality control system to have minimal variation<br>between lots. For this reason, lot information is not included in this table. Analysis was performed by an accompanied<br>software package MACSIQ View Analysis also described in the MACSima publication<br>(https://doi.org/10.1038/s41598-022-05841-4). The result of the analysis is included in the uploaded dataset. In<br>brief, the software processes the raw images of the MACSima run, generates stitched images, and then allows downstream<br>analysis including cell segmentation, cell gating, data normalization, dimension reduction plots (tSNE, UMAP), heat<br>maps, distance analyses and cluster analyses, all of which are interactively linked together. The MACSima system is<br>continuously evolving, this is the second OMAP for the MACSima system.