OMAP-23: Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of vermiform appendix (FFPE) with MICS on MACSima, v1.0

OMAP-23 was designed for MICS (MACSima imaging cyclic staining) imaging of FFPE human vermiform appendix<br>sample. Tissue fixation and antigen retrieval is described in (Spatial protein and RNA analysis on the same tissue<br>section using MICS technology). The MACSima technology is described in detail in the following publication (MACSima<br>imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid<br>tumors). All, antibodies in this panel are recombinant antibodies with a mutated human IgG1 constant region, removing<br>Fc receptor binding capacity of human IgG1, eliminating the need for additional blocking steps and reducing<br>non-specific binding. The use of human IgG1 recombinant antibodies allows for the addition of uncoupled monoclonal<br>antibodies from other species followed by a fluorescence labelled secondary reagent specific for the species of the<br>monoclonal antibody. The multiplex system has been described already for OMAP-10 and OMAP-21. A new dye VioB515 is<br>used for one reagents. The fluorescence is removed by cleavage. The panel contains 28 antibodies and the nuclear<br>marker DAPI for image alignment and nuclear segmentation. This OMAP provides a spatial context for all anatomical<br>structures and most cell types present in the vermiform appendix. OMAP-23 follows OMAP-21, with fewer antibodies but<br>adding antibodies for non-immune cells. All reagents are from Miltenyi Biotec and have been rigorously tested through<br>an internal quality control system to have minimal variation between lots. For this reason, lot information is not<br>included in this table. Analysis was performed by an accompanied software package MACSIQ View Analysis also described<br>in the MACSima publication (https://doi.org/10.1038/s41598-022-05841-4). The MACSima system is continuously evolving,<br> this is the third OMAP for the MACSima system.