OMAP-10: Multiplexed Antibody-Based Imaging of Human Palatine Tonsil with MACSima, v1.1

OMAP-10 was designed for MACSima (MACSima imaging cyclic staining) imaging of paraformaldehyde (PFA)-fixed human<br>tonsil samples. The MACSima technology is described in detail in the following publication<br>(https://doi.org/10.1038/s41598-022-05841-4). Most, but not all, antibodies in this panel are recombinant antibodies<br>with a mutated human IgG1 constant region. The described mutation removes the Fc receptor binding capacity of human<br>IgG1, eliminating the need for additional blocking steps and reducing non-specific binding of human antibodies on<br>human tissues. Highly multiplexed imaging is achieved through cycles of immunolabeling with FITC, PE, and APC<br>conjugated antibodies and photobleaching to eliminate fluorescence signal between imaging cycles. The panel contains<br>30 antibodies and the nuclear marker DAPI for image alignment and nuclear segmentation. This OMAP provides a spatial<br>context for all anatomical structures and most cell types present in the human palatine tonsil. OMAP-10 follows<br>closely OMAP-1 described for human lymph nodes<br>(https://cdn.humanatlas.io/hra-releases/v1.3/docs/omap/omap-1-human-lymph-node-ibex.html). All reagents were obtained<br>from Miltenyi Biotec and have been rigorously tested through an internal quality control system to have minimal<br>variation between lots. For this reason, lot information is not included in this table. Analysis was performed by an<br>accompanied 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, and this is the first OMAP for the MACSima system. A representative dataset created using<br>OMAP-10 can be found here: https://doi.org/10.5281/zenodo.7875937.