CODEX multiplexed tissue imaging with DNA-conjugated antibodies

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 similar to 4.5 h, validation of DNA-conjugated antibodies with CODEX staining takes similar to 6.5 h and preparation for a CODEX multicycle experiment takes similar to 8 h. The multicycle imaging and data analysis time depends on the tissue size, number of markers in the panel and computational complexity.

Automated summary

Advances in multiplexed imaging technologies, such as CODEX, using DNA-conjugated antibodies and fluorescently labeled DNA probes, allow visualization of up to 60 markers simultaneously in tissues. This enables a deep view into single-cell spatial relationships and can drive discovery in developmental biology, disease, and therapeutic design.