Current Methods in CODEX High-Multiplex Microscopy to Describe Spatial Relationships in the Tissue Microenvironment

Illuminating mechanisms at the tissue and cellular levels using indexed oligonucleotide-conjugated antibodies has further increased the resolution necessary to study pathological disease at microscopic levels. CO-Detection by indEXing (CODEX) is a high-multiplex microscopy method that uses up to 100 unique conjugated antibodies to elucidate which cell types and cellular neighborhoods are present in the imaged tissue, to determine their positions relative to each other and their functional states for physiologically-based mechanistic derivations. The first part of these procedures describes how to conjugate antibodies to their assigned oligonucleotide. Then, these conjugates are all optimized for either formalin-fixed paraffin-embedded (FFPE) or fresh-frozen (FF) tissues. In the third part, the conjugate-stained tissue section undergoes the CODEX multicycle reaction where fluorescently labeled oligonucleotides complementary to conjugates are periodically added and removed to aid in the capture of images. Lastly, the collected images are processed to set the stage for cell type and cellular neighborhood mapping on the imaged tissue for further spatial analysis work. This method has advanced basic antibody research and translational pathology workflows as well as refining new clinical treatments by providing bioinformatic support to clinicians to help patients with various diseases, especially cancer.