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Related Experiment Video

Updated: Sep 10, 2025

Optimized Workflow for Iterative Bleaching Extends Multiplexity Imaging of Highly Autofluorescent Clinical Samples

Optimized Workflow for Iterative Bleaching Extends Multiplexity Imaging of Highly Autofluorescent Clinical Samples

Published on: July 11, 2025

Iterative Bleaching Extends Multiplicity with Use of Staining Automation for Core Facilities.

Anna E Tseng¹, Aoife O'Connell², Aaron Benhamou³

¹National Emerging Infectious Diseases Laboratories (NEIDL), Boston University; Department of Pathology and Laboratory Medicine, Boston University Chobanian and Avedisian School of Medicine.

Journal of Visualized Experiments : Jove

|August 25, 2025

Summary

Automated Iterative Bleaching Extends Multiplicity (IBEX) enhances protein analysis in tissues. This tyramide signal amplification technique improves throughput and reproducibility for core facilities using autostainers.

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Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Published on: January 21, 2019

Area of Science:

Biomedical Imaging
Molecular Pathology
Immunohistochemistry

Background:

Iterative Bleaching Extends Multiplicity (IBEX) is a tyramide signal amplification (TSA)-based technique for multiplexed protein imaging in single tissue samples.
Current IBEX implementation often requires manual steps, limiting throughput and reproducibility.
Automation of IBEX in core facilities can significantly enhance efficiency and standardization.

Purpose of the Study:

To demonstrate the successful application of automated IBEX on common autostainer platforms.
To provide optimization strategies for designing IBEX panels for formalin-fixed, paraffin-embedded (FFPE) tissues.
To guide core pathology facilities in integrating automated IBEX into their workflows.

Main Methods:

Implementation of IBEX on two distinct autostainer platforms.
Development of methodologies for testing antibody-antigen retrieval sensitivity for panel design.
Utilizing whole-slide scanners for imaging and cell phenotyping software for analysis.

Main Results:

Successful application of automated IBEX on two autostainer platforms.
Detailed considerations for TSA-based panel design in FFPE samples, including antibody-antigen retrieval sensitivity testing.
Demonstration of downstream imaging and analysis using commercial whole-slide scanners and cell phenotyping software.

Conclusions:

Automated IBEX is versatile and can be readily integrated into existing core facility autostaining platforms.
The study provides practical guidance and methodologies for optimizing IBEX implementation and panel design.
Automation of IBEX offers increased throughput, reproducibility, and efficiency for multiplexed protein analysis.