These datasets were 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., 2020, doi.org/10.1073/pnas.2018488117. “IBEX: An open and extensible method for high content multiplex imaging of diverse tissues”, A. Radtke et al., 2021, arXiv:2107.11364. Acquisition of 3D data utilized the manual protocol while acquisition of the 2D data utilized the automated protocol. All data is stored using the Imaris file format. To view these multi-channel images, you can either use one of these free viewers, Imaris viewer (Oxford Instruments), Fiji. Images can be registered using the code available on GitHub (XTRegisterSameChannel: “Affine registration of z-stacks using common channel”). A video illustrating the usage of the Imaris extension to register this data is available on YouTube. Human Spleen Dataset is comprised of a four-cycle manual IBEX experiment performed on human spleen sections labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. 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 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 um), y (0.284 um), and z (1 um). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976). Markers per channel in each of the cycles: Human_Spleen_Panel1.ims (7 channels): CD21, CD54, Hoechst, CD20, CD68, Glycophorin A, Fibrinogen Human_Spleen_Panel2.ims (7 channels): CD163, CD49a, CD138, Hoechst, CD15, HLA-DR, CD11c Human_Spleen_Panel3.ims (7 channels): Vimentin, CD4, CD31, Ki-67, Hoechst, CD3, CD8 Human_Spleen_Panel4.ims (7 channels): SPARC, CD44, Hoechst, CD61, CD45, SMA, Lumican Registration - If using our Imaris Extensions registration code, this dataset uses the default settings. Human Liver Dataset is comprised of a four-cycle manual IBEX experiment performed on human liver sections labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. 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 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 um), y (0.284 um), and z (1 um). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976). Markers per channel in each of the cycles: Human_Liver_Panel1.ims (6 channels): CD54, ASS1, Lyve-1, Hoechst, Keratin 7, CD34 Human_Liver_Panel2.ims (6 channels): CD163, CD138, Fibrinogen, Hoechst, HLA-DR, CD68 Human_Liver_Panel3.ims (7 channels): CD4, Glutamine Synthetase, Ki-67, Hoechst, CD3, CD8, Tubulin beta 3 Human_Liver_Panel4.ims (6 channels): CD44, Vimentin, CD49a, Hoechst, SMA, CD45 Registration - If using our Imaris Extensions registration code, this dataset requires that you modify the default settings. Using the advanced settings dialog you need to: Disable the FFT option. Enable the 2D affine option. Human Kidney Dataset is comprised of a four-cycle automated IBEX experiment performed on human kidney Formalin-Fixed Paraffin-Embedded (FFPE) sections labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. Images were acquired using an inverted Thunder 3D Cell Culture microscope with a high precision (Quantum) stage, a high quantum efficiency sCMOS camera (DFC9000 GTC), and a 40x (1.3) NA oil objective. The light source was an LED8 with 8 individual LED lines for excitation with millisecond triggering. All images were captured at a 16-bit depth with the following pixel dimensions: x (0.160 um), y (0.160 um), and z (1 um). Images were tiled and merged using the LAS X Navigator software (LAS X 3.7.1.21655). Markers per channel in each of the cycles: Human_Kidney_Panel1.ims (4 channels): Hoechst, Cytokeratin, CD34, SMA Human_Kidney_Panel2.ims (3 channels): Hoechst, CD10, Cathepsin L Human_Kidney_Panel3.ims (3 channels): Hoechst, CD15, TMPRSS2 Human_Kidney_Panel4.ims (3 channels): Hoechst, Vimentin, Glycophorin A Registration - If using our Imaris Extensions registration code, this dataset uses the default settings.