CODEX Multiplexed Tissue Imaging with DNA-conjugated Antibodies

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2021 Jul 3

PMID 34215862

Citations 206

Authors

Sarah Black

Darci Phillips

John W Hickey

Julia Kennedy-Darling

Vishal G Venkataraaman

Nikolay Samusik

Yury Goltsev

Christian M Schurch

Garry P Nolan

Affiliations

Soon will be listed here.

Abstract

Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 60 markers in situ. CODEX enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in developmental biology, disease and therapeutic design. Herein, we provide optimized protocols for conjugating purified antibodies to DNA oligonucleotides, validating the conjugation by CODEX staining and executing the CODEX multicycle imaging procedure for both formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissues. In addition, we describe basic image processing and data analysis procedures. We apply this approach to an FFPE human tonsil multicycle experiment. The hands-on experimental time for antibody conjugation is ~4.5 h, validation of DNA-conjugated antibodies with CODEX staining takes ~6.5 h and preparation for a CODEX multicycle experiment takes ~8 h. The multicycle imaging and data analysis time depends on the tissue size, number of markers in the panel and computational complexity.

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