Fluorescence microscopy is typically limited to 4-5 separate color channels, but cell type identification, especially in neuroscience and immunology, depends on simultaneously measuring >10 antigens per cell. We have onboarded and tested technologies seeking to increase the plex of spatial proteomics, including CODEX, MIBI, ImmunoSABER/Ultivue, and Nanostring GeoMx. Of these, CODEX, or CO-detection by inDEXing, offered the best balance of throughput, reliability, and customizability (Goltsev et al., 2018; Schürch et al., 2019). Our custom implementation uses automated fluidics from Akoya Biosciences running on an Andor Dragonfly 200 spinning disk confocal microscope, which enables high resolution full coverslip imaging (18 x 18 mm) over 40 channels in 24 hours and removes the need for z-stack deconvolution. We have tested this configuration across >15 tissue types, spanning mouse, human (FFPE and OCT) in imaging experiments detecting up to 40 antigens.
Highly multiplexed immunostaining
