Halo 3

Single-marker IHC images were uploaded into HALO 3.3 image analysis software (Indica Labs, Corrales, NM). Boundaries of each anatomical region of interest (ErC, CA1-ProS border region, gray matter of middle temporal gyrus, pontine LC plus raphe nucleus) were identified and annotated using a combination of anatomical landmarks (rhinal sulcus, dentate gyrus, temporal horn of lateral ventricle, pyramidal blades of the dentate gyrus, 4th ventricle) and cytoarchitectonic mapping as previously described.(46 (link)) MP-IHC with immunolabeling of numerous cytoarchitectural markers was performed on serial sections to assist with identification of anatomical landmarks and boundaries including white matter tracts and gray matter-white matter interfaces as previously described. (46 (link)) Stain positive area as a percentage of each annotated region was quantified using the HALO Area Quantification v2.2.1 module. Plaque-associated objects per mm² within each annotated region were identified and quantified using the HALO Object Colocalization v1.3 module with classifier function enabled as previously described.(46 (link))

Single-marker IHC images were uploaded into HALO 3.3 image analysis software (Indica Labs, Corrales, NM). Boundaries of each anatomical region of interest (ErC, CA1-ProS border region, gray matter of middle temporal gyrus, pontine LC plus raphe nucleus) were identified and annotated using a combination of anatomical landmarks (rhinal sulcus, dentate gyrus, temporal horn of lateral ventricle, pyramidal blades of the dentate gyrus, 4^th ventricle) and cytoarchitectonic mapping as previously described.^{46 (link)} MP-IHC with immunolabeling of numerous cytoarchitectural markers was performed on serial sections to assist with identification of anatomical landmarks and boundaries including white matter tracts and gray matter-white matter interfaces as previously described.^{46 (link)} Stain positive area as a percentage of each annotated region was quantified using the HALO Area Quantification v2.2.1 module. Plaque-associated objects per mm² within each annotated region were identified and quantified using the HALO Object Colocalization v1.3 module with classifier function enabled as previously described.^{46 (link)}

FFPE sections, 4-µm thick, were subjected to staining using an automated staining system (BOND-MAX; Leica Microsystems) with the 6-color Kit (Abcarta) following the manufacturer's instructions. The markers were grouped into two distinct panels: a macrophage panel and a T-cell panel. The macrophage panel included SOX10 (EP268, ZSGB-BIO), CD68 (KP1, MXB Biotechnologies), CD163 (10D6, ZSGB-BIO), CD80 (ab134120, Abcam), and CD206 (ab64693, Abcam). T-cell panel consisted of SOX10, CD4 (458G4A1, Abcarta), CD8 (815R4B2, Abcarta), CD69 (ab233396, Abcam), and CD45RO (UCHL-1, MXB Biotechnologies). Normal human tonsil FFPE tissues were also stained with and without primary antibodies to serve as positive and negative controls, respectively. All stained slides were scanned using a digital pathology slide scanner (× 40, KF-PRO-005, KFBIO). Trained pathologists utilized image analysis software (HALO 3.3, Indica Labs) to discern and quantify cell phenotypes. Moreover, each case was subject to manual review to ensure consistency and accuracy. The positive rate of immune cells is defined as the proportion of immune cells to total cells. In addition, the proportion of M1 or M2 macrophages to the total number of macrophages is used for comparison between PMME and NEMM.