OMAP-21: Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of Human Palatine Tonsil with MICS on MACSima 1.5

OMAP-21 was designed for MICS  (MACSima imaging cyclic staining) imaging of FFPE human tonsil samples.  Tissue fixation and antigen retrieval is described in (Spatial protein and RNA analysis on the same tissue section using MICS technology). The MACSima technology is described in detail in the following publication (MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors). Most, but not all, antibodies in this panel are recombinant antibodies with a mutated human IgG1 constant region. The described mutation removes the Fc receptor binding capacity of human IgG1, eliminating the need for additional blocking steps and reducing non-specific binding of human antibodies on human tissues. Highly multiplexed imaging is achieved through cycles of immunolabeling with FITC, PE, and APC conjugated antibodies and photobleaching to eliminate fluorescence signal between imaging cycles. The panel contains 47 antibodies and the nuclear marker DAPI for image alignment and nuclear segmentation. This OMAP provides a spatial context for all anatomical structures and most cell types present in the human palatine tonsil. OMAP-21 follows closely OMAP-1 described for human lymph nodes (omap-1-human-lymph-node-ibex) and OMAP-10 (omap-10-palatine-tonsil-macsima).

All reagents were obtained from Miltenyi Biotec and have been rigorously tested through an internal quality control system to have minimal variation between lots. For this reason, lot information is not included in this table. Analysis was performed by an accompanied software package MACSIQ View Analysis also described in the MACSima publication (https://doi.org/10.1038/s41598-022-05841-4). The result of the analysis is included in the uploaded dataset. In brief, the software processes the raw images of the MACSima run, generates stitched images, and then allows downstream analysis including cell segmentation, cell gating, data normalization, dimension reduction plots (tSNE, UMAP), heat maps, distance analyses and cluster analyses, all of which are interactively linked together. The MACSima system is continuously evolving,  this is the second OMAP for the MACSima system. A representative dataset created using OMAP-21 can be found here:  10.5281/zenodo.11281609 .