Fig 2: ICS increased TH, DAT, and DA levels and decreased GFAP, Iba1, and α-syn levels in striatum of METH mice model. Figures 3(a)–3(c) Representative WB images and quantification of TH and DAT. (d) Measurement of DA levels by using HPLC. Figures 3(e)–3(h) Representative WB images and quantification of GFAP, Iba1, and α-syn. n = 4 per group.

Fig 3: Dynamic change in Siglec-15 expression in TAMs in human glioma and GL261 murine glioma assessed by immunofluorescence staining. Left graph: (A) Representative images and Optical density (OD) score of Siglec-15 expression in human glioma tissues from grade I to grade IV. (B–D) Representative images and OD score of Siglec-15, CD68, TMEM119 and Iba1 in allografts tissue on the indicated days after tumor implantation. C57BL/6 mice were intracranially injected with GL261 cells, tumor tissues were collected on (D7, D14, D21, D28) to staining target protein. Siglec-15 and CD68 were stained by immunofluorescence staining (B, C), and TMEM119 and Iba1 were stained by IHC (D). Right graph: OD scores of target gene expression were quantified by ImageJ software. Scale bar: 250 μm, original magnification 40×; 100 μm, original magnification 100×; 50 μm, original magnification 200×; 25 μm, original magnification 400×. Data are representative of three independent experiments. *** P< 0.001; **** P< 0.0001.

Fig 4: Spatial analysis of microglia in Alzheimer’s disease alters their cellular state according to proximity to amyloid plaques.(A) Segmentation of amyloid plaques (left) and tau tangles (right) from one human AD hippocampus by eZsegmenter through masking (blue) Pan-Aβ (magenta) and PHF-tau (yellow) expression. Human donor details: 82-year-old male with AD dementia, Braak score of V, MMSE score of 19, APOE ε3/ε3, and a post-mortem interval of 2.95 hours.(B) Phenotyping of plaque types with FlowSOM clustering (left, top) results in four plaque types with varying levels of Pan-Aβ, Aβ42 and Aβ40. Density of each plaque type quantified as number of plaques per mm2 in the grey and deep white matter of the human AD hippocampus (left, bottom). Relative levels of each plaque phenotyping protein for each plaque type (right).(C) Spatial enrichment analysis of plaques and tangles proximal to single microglia (left) through masking each feature and quantifying the presence of a plaque or tangle within a specified radius (r = major axis length of each cell + 10 pixel buffer) around each microglial mask. Total number of microglia with a plaque or tangle of each type within their direct vicinity (right).(D) Quantification of differentially expressed proteins in disease associated microglia (DAMs) as defined by their proximity to each plaque type, with representative plaque types 3 and 4 shown with microglia overlaid as dashed magenta lines.(E) Microglial cellular density along the MSC between the healthy (green line) and AD (navy blue line) human hippocampus (top) with the total number of statistically significant and not significant pairwise differences within protein and morphology features.(F) Statistically significant feature differences (only proteins) between the healthy and AD human hippocampus as volcano plot, with their PCTBP scores and area between the curves.(G) All feature comparisons (protein and morphology) shown along the MSC for the healthy and AD human hippocampi.(H) Validation of MIBI findings through an independent cohort of 13 healthy and 13 AD hippocampi through low-plex immunohistochemistry: (top) staining the tissue microarray cores of the CA1 region of the hippocampus with Iba1 (blue) and HLA-DR (brown) as a dual stain and in silico separation of each protein signal; (bottom) segmentation of microglia through masking both Iba1 and HLA-DR signals together in eZsegmenter and quantification of signal intensity at a single-cell level in each FOV acquired in healthy and AD hippocampus groups. A proxy MSC was calculated with Iba1 and HLA-DR expression from extracted cells (bottom, middle) and the average microglial cellular density plotted for the healthy (green line) and AD (navy blue line) hippocampus (with individual donors in dashed lines) and Iba1 (light blue) and HLA-DR (peach) expression along the MSC. Statistical significance (p-value) was calculated along binned MSCs (bottom, right) with PCTBP scores > 1 interpreted as statistically significant.

Fig 5: Microglial segmentation and phenotyping within the context of large anatomical brain regions.(A) Iba1+ (green) microglia depicted in a single FOV from the human hippocampus in a < 1 mm depth of field, with nuclear staining by histone H3 (HH3, blue) and vascular staining by CD31 + CD105 (red).(B) An enlarged view of FOV1 in (A) highlighting a microglia cell (Cells 1) positive for Iba1 (green) and CD45 (red) with its HH3+ (blue) nucleus in the plane.(C) Microglia were segmented with eZsegmenter by creating a hybrid mask (green) based on Iba1 (red) and CD45 (blue) expression, normalized for cell size.(D) Inclusion of microglial nuclei (HH3, blue) from the segmentation mask and exclusion of other glial markers like GFAP (red) and S100β (blue).(E) Microglial cellular density in local expert-annotated sub-regions across the hippocampus and cerebellum, depicted as color overlay on dots (microglia) across each stitched image.(F) Quantification of local microglial density in each acquired FOV for each large anatomical brain regions in grey and deep white matter areas, including the hippocampus (HIP), substantia nigra (SN), middle frontal gyrus (MFG), caudate and cerebellum (n = 1 for each brain region). Box and whisker plots represent the minimum, first quantile, median, third quantile and maximum, with individual points representing FOVs.(G) Microglial phenotyping protein expression within segmented cellular masks across five sets of single cells showing differential intracellular localization of each protein.(H) Endogenous elemental iron (Fe) found within and outside of segmented microglia and its colocalization with Ferritin-L.(I) Pearson correlation of microglial phenotyping proteins at a cellular level, with specific positively and negatively correlated protein programs highlighted.