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Related Experiment Video

Updated: Oct 30, 2025

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples
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CODEX multiplexed tissue imaging with DNA-conjugated antibodies.

Sarah Black1,2, Darci Phillips1,2, John W Hickey1,2

  • 1Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA, USA.

Nature Protocols
|July 3, 2021
PubMed
Summary

This study optimizes Co-detection by indexing (CODEX) protocols for multiplexed imaging, enabling detailed single-cell analysis in tissues. The optimized methods facilitate antibody conjugation and multicycle imaging for enhanced biological discovery.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Cell Biology

Background:

  • Multiplexed imaging advances single-cell tissue characterization.
  • Co-detection by indexing (CODEX) visualizes up to 60 markers in situ using DNA-conjugated antibodies.
  • CODEX provides insights into single-cell spatial relationships for biological discovery.

Purpose of the Study:

  • To provide optimized protocols for antibody conjugation to DNA oligonucleotides.
  • To detail CODEX multicycle imaging procedures for FFPE and fresh-frozen tissues.
  • To describe basic image processing and data analysis for CODEX experiments.

Main Methods:

  • Antibody conjugation to DNA oligonucleotides.
  • CODEX staining for conjugation validation.
  • Multicycle imaging for formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissues.
  • Image processing and data analysis.

Main Results:

  • Optimized protocols for antibody conjugation and CODEX staining are presented.
  • Successful application of CODEX to FFPE human tonsil multicycle imaging.
  • Experimental timeframes provided for antibody conjugation, validation, and experiment preparation.

Conclusions:

  • Optimized CODEX protocols enhance single-cell spatial analysis capabilities.
  • The described methods facilitate in situ visualization of numerous markers in diverse tissue types.
  • This work supports advancements in developmental biology, disease research, and therapeutic design.