Multiplex immunofluorescence staining, tissue imaging and cell phenotyping
A validated and standardized multiplex immunofluorescence protocol was developed for simultaneous detection of CD3, CD8, CD11b, CD163, GFAP and DAPI in a single FFPE tissue section. The validation pipeline for the multiplex immunofluorescence protocol has been previously described by our groupMultiplexed immunofluorescence slides were scanned on a Vectra-Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences). Spectral unmixing was performed using inForm software (version 2.4.8, Akoya Biosciences), as described. Image analysis was performed using QuPath and Fiji/ImageJ. Briefly, cells were segmented on the basis of nuclear detection using the StarDist 2D algorithm. A random trees algorithm classifier was trained for each cell marker. Cells were then subclassified as CD3