Ascending high-plex mountains with IBEX: An open and versatile method for multiplexed antibody-based imaging

IBEX is an iterative immunolabeling and chemical bleaching method that enables highly multiplexed imaging in diverse tissues. IBEX is compatible with over 250 commercially available antibodies, 16 unique fluorophores, and can be easily adopted to different imaging platforms.
Published in Protocols & Methods
Ascending high-plex mountains with IBEX: An open and versatile method for multiplexed antibody-based imaging

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Life can only be understood backwards, but it must be lived forwards. ~ Soren Kierkegaard

While pursuing graduate research in medical entomology, I attended a seminar showcasing live imaging of Leishmania parasites in the skin1. I was captivated! This was my introduction to the work of the Germain laboratory. Several years passed without my thinking of imaging until I was tasked with teaching myself immunofluorescence microscopy. Failure teaches success. Over 8 months, I received a thorough education in failure with modest success until fate intervened. I was introduced to Wolfgang Kastenmüller, a post-doctoral fellow in the Germain laboratory, by his wife Kathrin, a collaborator with my PhD advisor Fidel Zavala. The Kastenmüllers were extraordinarily helpful. Wolfgang, an imaging virtuoso, shared his extensive knowledge of static and dynamic imaging. We laughed upon finding that we share a birthday and nickname. Wolfgang ultimately introduced me to Ron Germain who was, and continues to be, generous with his wisdom and resources. Following the completion of my PhD, I joined the Germain lab as a post-doctoral fellow where I have worked beside some exceptional immunologists and microscopists. Of note, Michael Gerner led us all in hyperspectral imaging and histo-cytometry2. Without question, the IBEX method detailed here3 rests on the shoulders of Germain imaging giants. 

For the strength of the pack is the wolf, and the strength of the wolf is the pack. ~ Rudyard Kipling

My colleagues and I have built upon this foundation by developing IBEX, an open-source multiplexed antibody-based imaging method that allows dozens of protein biomarkers to be visualized in a single tissue section4 (Figure 1). To aid these efforts, we developed a tissue fixation method that preserves tissue architecture, minimizes autofluorescence, and is compatible with hundreds of mouse and human antibodies (Table). We worked closely with surgeons and pathologists (Drs. Jeremy Davis, Jonathan Hernandez, Danny Jonigk, and Stefania Pittaluga) to create procedures for grossing tissues in support of a Human Reference Atlas5-7. These details and others are captured in the figures designed by graphical artist Li Yao ( To develop the original method4, my student Evelyn Kandov and I had to overcome several technical challenges as well as limitations with existing iterative imaging protocols. The obstacle is the way. After months of troubleshooting, we developed the manual IBEX protocol which achieves tissue adherence using chrome gelatin alum, accelerates antibody immunolabeling with a non-heating microwave, and streamlines fluorophore inactivation using lithium borohydride. To date, the IBEX method has been performed hundreds of times by dozens of colleagues both within and outside of the Germain lab, including Anita Gola, Hiroshi Ichise, Rochelle Shih, and efforts organized by Joshua Croteau. Here, we expand on key details for safe and successful adoption of the method. To capitalize on the marker depth provided by iterative imaging, Ziv Yaniv and Brad Lowekamp built software tools for image alignment using SimpleITK8,9. The work of Ziv, Brad, and Juraj Kabat evolved the process from tedious and error-prone, e.g. typos, walking files across campus aka ‘Sneakernet’, to streamlined and accessible to all ( In keeping with broader implementation and ease, Colin Chu and James Marr automated IBEX using a fluidics device and widefield microscope, resulting in a cost-effective solution for mapping tissues at single cell resolution.

A map, it is said, organizes wonder. ~ Ellen Meloy

Maps bring order to the dazzling world around us10-12, and there’s no greater terrain than the diverse cellular ecosystems present in normal and diseased tissues. With the details included here, IBEX can be applied to a variety of questions: from atlas building efforts6,7,13 to mechanistic studies involving experimental animal models14. Beyond supporting manual and automated IBEX imaging, the extensive material list and detailed instructions assembled here are a great resource for others interested in obtaining high quality imaging data using traditional immunofluorescence or other multiplexed antibody-based imaging methods15 (Table). To this end, we are delighted to see our microwave labeling program used for three-dimensional (3D) immunofluorescence microscopy16, and Emily Speranza’s Opal-plex, IBEX imaging of heavily fixed tissues with Opal fluorophores, applied to other biological questions17.

High in the mountains live the wild goats. ~ Psalm 104:18 (NLT)

Whether one adopts some or all aspects of IBEX (Table), my co-authors and I wish everyone the success and sure-footed determination of the method’s namesake (Figure 2). These gravity-defying creatures are adept at climbing sheer rockfaces in the quest for survival. Science can feel a lot like that. While the profound symbolism of ibex was always known to us, it’s true significance would be revealed several years later. Our original IBEX manuscript was born a Capricorn, appearing online on the auspicious winter solstice and in print December 29th, 20204. We are delighted to celebrate another Capricorn birthday with a publication date of January 12th, 2022 in Nature Protocols. For years we searched for a fluorophore with the spectral properties of Alexa Fluor 594 that could be inactivated within 15 minutes of exposure to borohydride compounds. Rebecca Beuschel and I tried several alternatives before fate introduced us to Tanja Guest of Caprico Biotechnologies. Tanja recommended iFluor 594, an excellent conjugate and now staple of our IBEX imaging panels. In closing, we hope IBEX allows a greater understanding of tissue landscapes from the elevated vistas only multiplexed imaging can provide. We are particularly encouraged by the budding developer community and look forward to building an open platform that includes robust image analysis (Nishant Thakur and Spencer Grant, manuscript in preparation; Ed Schrom, in progress).

Welcome wild goats!

Figure 1. Highly multiplexed imaging of diverse mouse tissues using IBEX. 

(a) Confocal images from an immunized mouse LN (34 of 41 parameters shown). Scale bar is 200 µm (left), 50 µm (insets). (bConfocal images from mouse thymus (22 of 27 parameters shown). Scale bar is 200 µm (left), 20 µm (insets). Gamma Delta T cells (TCRgd) (c) Confocal images from mouse spleen (12 of 16 parameters shown). Scale bar is 50 µm (left), 20 µm (insets). (dConfocal images from mouse small intestine (16 of 20 parameters shown). Scale bar is 200 µm (left), 25 µm (insets). (e) Confocal images from mouse lung (15 of 23 parameters shown). Scale bar is 100 µm (left), 25 µm (insets). β-tubulin 3 (βtub3), Collagen IV (Coll IV). (f) Confocal images from mouse liver (12 of 18 parameters shown). Scale bar is 50 µm (left), 20 µm (insets). Glutamine synthetase (GS). Images acquired by Evelyn Kandov, Anita Gola, and Andrea Radtke. Please see for IBEX imaging of human tissues.

Table. Overview of technical information included in IBEX protocol.

The materials and methods outlined in this protocol are relevant for workflows beyond IBEX including traditional, single cycle immunofluorescence (IF) and immunohistochemistry (IHC) and other high content iterative methods. NA (not applicable).

Figure 2. Alpine ibex near the Black Lake "Lago Nero" in Valsesia (Italy). Photo credit: Anita Gola, a G.O.A.T. friend!


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14        Gola, A. et al. Commensal-driven immune zonation of the liver promotes host defence. Nature 589, 131-136, doi:10.1038/s41586-020-2977-2 (2021).

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