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.

多重成像技术的进步极大地提高了我们在单细胞水平上表征健康和患病组织的能力。索引共检测 (CODEX) 依赖于 DNA 偶联抗体和互补荧光标记 DNA 探针的循环添加和去除，迄今为止已被用于在原位同时可视化多达 60 个标记。CODEX 可以深入了解组织中的单细胞空间关系，旨在促进发育生物学、疾病和治疗设计方面的发现。在此，我们提供优化方案，用于将纯化的抗体与 DNA 寡核苷酸结合，通过 CODEX 染色验证结合，并为福尔马林固定、石蜡包埋 (FFPE) 和新鲜冷冻组织执行 CODEX 多循环成像程序。此外，我们描述了基本的图像处理和数据分析程序。我们将这种方法应用于 FFPE 人体扁桃体多周期实验。抗体偶联的动手实验时间约为 4.5 小时，使用 CODEX 染色验证 DNA 偶联抗体需要 ~6.5 小时，CODEX 多周期实验的准备时间约为 8 小时。多周期成像和数据分析时间取决于组织大小、面板中标记的数量和计算复杂性。