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

Abstract

High content imaging is needed to catalogue the variety of cellular phenotypes and multi-cellular ecosystems present in metazoan tissues. We recently developed Iterative Bleaching Extends multi-pleXity (IBEX), an iterative immunolabeling and chemical bleaching method that enables multiplexed imaging (>65 parameters) in diverse tissues, including human organs relevant for international consortia efforts. IBEX is compatible with over 250 commercially available antibodies, 16 unique fluorophores, and can be easily adopted to different imaging platforms using slides and non-proprietary imaging chambers. The overall protocol consists of iterative cycles of antibody labelling, imaging, and chemical bleaching that can be completed at relatively low cost in 2-5 days by biologists with basic laboratory skills. To support widespread adoption, we provide extensive details on tissue processing, curated lists of validated antibodies, and tissue-specific panels for multiplex imaging. Furthermore, instructions are included on how to automate the method using competitively priced instruments and reagents. Finally, we present a software solution for image alignment that can be executed by individuals without programming experience using open source software and freeware. In summary, IBEX is an open and versatile method that can be readily implemented by academic laboratories and scaled to achieve high content mapping of diverse tissues in support of a Human Reference Atlas or other such applications.

Robust, persistent adaptive immune responses to SARS-CoV-2 in the oropharyngeal lymphoid tissue of children

“…Sections were permeabilized, blocked, and stained in PBS containing 0.3% Triton X-100 (Sigma-Aldrich), 1% bovine serum albumin (Sigma-Aldrich), and 1% human Fc block (BD Biosciences). Immunolabeling was performed with the PELCO BioWave Pro 36500-230 microwave equipped with a PELCO SteadyTemp Pro 50062 Thermoelectric Recirculating Chiller (Ted Pella) using a 2-1-2-1-2-1-2-1-2 program 80,81 . A complete list of antibodies and imaging panels with labelling steps can be found in Supplementary Table 8.…”

Understanding immunity in a tissue‐centric context: Combining novel imaging methods and mathematics to extract new insights into function and dysfunction*

“…We are addressing both of these challenges with the development of Spatial Analysis of Cellular Ensembles (SPACE), a new analytical platform (Figure 3). For clear illustration, we use a 2‐dimensional static slice of human lymph node with just 8 markers, selected from the original image collected with over 60 markers using IBEX technology 11,92 and classified into 14 cellular phenotypes at the pixel level using RAPID‐P software (Figures 2D,E and 3A). However, SPACE runs efficiently on much larger and higher plex images.…”

“…Box 2 reveals the pericentral zone of the liver and liver sinusoidal endothelial cells (CD54, Lyve‐1), liver macrophages (CD163, HLA‐DR, Lyve‐1), and sinusoidal deposition of fibrinogen. Images are composed from original data published previously 11,92 …”

“…To aid in widespread adoption of the IBEX method, we recently authored a detailed protocol with instructions on how to automate data collection using a widefield microscope and cost‐effective fluidics device 92 . Compared with other imaging modalities that may require 27 hours to acquire a 1‐mm 2 area with a pixel size of 0.260 µm, 77 the automated IBEX method captures large regions of interest (>9 mm 2 ) at high optical resolution (0.160 µm in x‐y) in 14 hours with minimal (<1 hour) hands on time (24‐plex, 6 cycle, single z‐slice).…”

“…To aid in widespread adoption of the IBEX method, we recently authored a detailed protocol with instructions on how to automate data collection using a widefield microscope and cost-effective fluidics device. 92 Compared with other imaging modalities that may require 27 hours to acquire a 1-mm 2 area with a pixel size of 0.260 µm, 77 the automated IBEX method captures large regions of interest (>9 mm 2 ) at high optical resolution (0.160 µm in x-y) in Beyond developing a simple, cost-effective solution for multiplexed antibody-based imaging, the basic IBEX workflow has been extended to detect low-abundance epitopes in conventionally fixed tissues, for example, endogenous levels of the chemokine CXCL9 in the murine liver, 11,42 as well as image heavily fixed tissues from hosts infected with select agents such as the Ebola 11 and SARS-CoV-2 viruses. 93 The resulting method, Opal-plex, uses an unconjugated primary antibody, amplification with a horseradish peroxidase 11,92 cells in animal models of vaccination 42 along with detailed characterization of mouse and human stromal markers.…”