OMAP-10: Multiplexed Antibody-Based Imaging of Human Palatine Tonsil with MACSima, v1.2

OMAP-10 was designed for MICS (MACSima imaging cyclic staining) imaging of paraformaldehyde (PFA)-fixed human tonsil<br>samples. The MICS technology is described in detail in Kinkhabwala et al. (2022). Most antibodies in this panel are<br>recombinant antibodies with a mutated human IgG1 constant region that eliminates Fc receptor binding, reducing<br>non-specific binding and removing the need for blocking steps. Highly multiplexed imaging is achieved through cycles<br>of immunolabeling with FITC, PE, and APC conjugated antibodies followed by photobleaching between cycles. The panel<br>contains 30 antibodies and the nuclear marker DAPI for image alignment and nuclear segmentation. This OMAP provides<br>spatial context for all anatomical structures and most cell types in the human palatine tonsil. It follows closely the<br>antibody panel and methodology described in OMAP-1 for human lymph nodes. All reagents were obtained from Miltenyi<br>Biotec and were quality controlled internally; lot numbers are not provided. Image processing and downstream analysis<br>were conducted using the MACSIQ View Analysis software described in Kinkhabwala et al. (2022), which supports<br>segmentation, gating, normalization, dimensionality reduction (tSNE, UMAP), clustering, and spatial analyses with<br>interactive visualization. This is the first OMAP generated using the MACSima platform. A representative dataset is<br>available via Müller, Rüberg, and Bosio (2023).<br><br>**Bibliography:**<br><br>* Kinkhabwala, Ali, Christoph Herbel, Jennifer Pankratz, Dmytro A. Yushchenko, Silvia Rüberg, Paurush Praveen, Sandy Reiß, et al. 2022. “MACSima Imaging Cyclic Staining (MICS) Technology Reveals Combinatorial Target Pairs for CAR T Cell Treatment of Solid Tumors.” *Scientific Reports* 12 (1): 1911. https://doi.org/10.1038/s41598-022-05841-4.<br>* Müller, Werner, Silvia Rüberg, and Andreas Bosio. 2023. “OMAP-10: Multiplexed Antibody-Based Imaging of Human Palatine Tonsil with MACSima v1.0.” Zenodo. https://zenodo.org/records/7875938.