Fig 2: In situ confirmation of the abundance of major microglial subsets.a RNA expression levels of markers enriched in different subsets of microglia in the scRNA-seq dataset: ISG15 for interferon cluster 4, CD83 for the cytokine signaling enriched clusters 5 and 6, CD74 for antigen presentation related cluster 7 and PCNA for the proliferative cluster 9. The size of the circles represent the percentage of cells per cluster in which the given gene was detected, while the color coding represent the normalized z-scores. The z-score matrix is available in Supplementary Data 16. b Box-and-whisker plot representing the normalized gene expression of CD74 (in TPMs) among the different microglia clusters. Note that CD74 gene expression is ~2-fold higher in cluster 7 when compared to the expression in other clusters (see Source Data file). The boxes represent the 25th percentile, median, and 75th percentile. The whiskers extend to the furthest value that is no more than 1.5 times the inter-quartile range (default parameter for R’s boxplot function). The number of cells in each microglial cluster is also shown. c Distribution of the expression levels of CD74 on microglia in situ as measured by immunofluorescence and CellProfiler analysis. Note the second small peak at high expression values that we highlight with a red box. Source data are provided in the Source Data file. d Black symbols represent the quantification of the ISG15+, CD83+, and PCNA+ and CD74high microglia in the dorsolateral prefrontal cortex of seven individuals of mixed neuropathology (see Supplementary Data 9). The orange symbols represent the proportions for each subset observed in the single-cell RNA sequencing data. Center line represents the mean. Source data are available in the Source Data file. e Photomicrographs showing representative cells expressing the markers of the different microglia subsets. The arrows point to representative cells for each marker that are shown in the higher magnification photomicrographs in the far right column. In the micrographs showing CD74 staining, arrowhead points to a CD74 dim cell, while the arrow points to a CD74 bright (or high) cell. The bar in the lower right corner micrograph represents 100 µm for the overview images. The bar in the lower right corner of the higher magnification images (right most column) represents 50 µm. These experiments were performed in seven individual donors. In each donor 15–20 images were captured in the gray matter of the DLPFC and analyzed using IHC and automated image analysis. TPM transcripts per million.