OMAP-19 Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of Human Tonsil with IBEX

Representative dataset acquired using the Iterative Bleaching Extends multi-pleXity (IBEX) imaging method described in:

“IBEX: A versatile multi-plex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues“, A. Radtke et al., Proc. Natl. Acad. Sci. USA, 2020, doi.org/10.1073/pnas.2018488117.

"IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues", A. J. Radtke, et al., Nat Protoc, 2022, doi.org/10.1038/s41596-021-00644-9.

This dataset accompanies the Organ Mapping Antibody Panel (OMAP) effort led by the Human BioMolecular Atlas Program detailed here.

OMAP-19 was designed for IBEX imaging of human FFPE tonsil samples sectioned onto glass slides. The panel consists of 6 cycles of 20 primary antibodies, 5 secondary antibodies, and the nuclear label Hoechst for image alignment and nuclear segmentation. This OMAP provides a spatial context for 10 anatomical structures and at least 16 cell types present in the ASCT+B tonsil table. OMAP-19 was designed to examine myeloid subsets (CD11b, CD11c, CD14, CD15, HLA-DR) and angiogenesis (VEGF-A and VEGF-C) in human ovarian cancer biopsies. The human tonsil was used as a positive control for antibody validation and panel development. Antigen retrieval was performed using a pressure cooker (Borg Decloaker BD1000, 110°C for 15 minutes) and a tris-based buffer (pH 9.5). Several custom antibodies were created using commercial labeling kits as indicated in the associated RRID entries for these antibodies. More details on the antigen retrieval protocol and step-by-step application of antibodies can be found on the IBEX Imaging Community knowlege-base (current version) and on Zenodo (last official version).

Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as a 405 nm laser. All images were captured at an 16-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.379 µm), y (0.379 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software.

Image supplied as a .ims Imaris Format file and can be opened with the free Imaris Viewer software or ImageJ/Fiji. Image channel and antibody meta-data supplied as an xlsx file.