Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging

IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues

“…While both IBEX methods are compatible with indirect immunolabeling, we prefer to use directly labeled antibodies from vendors or conjugated in house using commercial kits. In addition to the points raised by Hickey and colleagues 19 , we recommend purchasing >100 µg of antibody in a suitable format for conjugation; concentration of 1 mg/ml or greater in a buffer without carrier proteins or additives. For panels requiring multiple unconjugated antibodies, we advise placing these antibodies in earlier cycles to avoid unwanted detection by the secondary antibody with directly conjugated antibodies generated in the same host.…”

“…A central tenet of IBEX, and other such methods [4][5][6][7][8][9][11][12][13][14][15] , is the collection of reproducible multiplexed imaging data using antibodies specific for their intended targets. Recently, members of multiple tissue-mapping consortia including the HCA 1 , HuBMAP 2 , Human Tumor Atlas Network (HTAN) 47 , and Human Protein Atlas 48 , established guidelines for multiplexed antibody-based imaging methods 19 . We refer the reader to this work as it documents how to properly validate antibodies, build and test multiplexed antibody panels, conjugate custom antibody reagents, and overcome tissue autofluorescence.…”

“…The work outlined here and previously 23 offer a resource to the field of antibody-based imaging by providing exhaustive lists of validated reagents and approved panels for mouse and human tissues. It is our intent to enable high quality data generation while defraying the substantial cost, many thousands of US dollars for a 50-plex panel, and time, around 6 months, to develop equivalent panels de novo 19 . The primary limitation of the original IBEX method was that it required manual execution; however, we now have an affordable automated solution for high content imaging (Table S4).…”

“…To this end, several multiplexed antibody-based imaging methods have been developed [4][5][6][7][8][9][10][11][12][13][14][15] and reviewed [16][17][18] . Furthermore, a detailed comparison of these methods can be found in a recent commentary authored by domain experts in the field of spatial proteomics 19 . The majority of these existing methods generate high dimensional datasets through an iterative multistep process (a cycle) that includes: i) immunolabeling with antibodies, ii) image acquisition, and iii) fluorophore inactivation or antibody/chromogen removal.…”

“…Using commercially available reagents and microscopes, IBEX has been used to spatially characterize complex phenotypes in tissues from experimental animal models 24 as well as clinically relevant human samples 23 . Because antibody validation and panel development can be costly in terms of time and capital 19 , we provide curated lists of antibodies as well as example panels for several tissues. The majority of these resources are optimized for a fixed frozen method of tissue preservation that overcomes the need for antigen retrieval; however, we demonstrate how IBEX can be adopted to formalin-fixed, paraffin embedded (FFPE) tissues.…”