Fig. 2

Mapping Aβ aggregation and microglial reactivity in 5XFAD mouse brains. Brains from aged 5XFAD mice (6.5 months) and non-carrier (NC) controls were immunostained and cleared with iDISCO+. Aβ plaques (anti-6E10) and microglia (anti-iba1) were labeled in the left hemispheres. Brains were imaged in 3D with LSFM and analyzed using UNRAVEL. (A) Examples of raw immunoreactivity are shown with the dashed outlines representing the surface of the registered atlas (white). The scale bar in the zoomed inset is 100 µm. Ilastik segmentations are shown for Aβ plaques and reactive microglia for 5XFAD mice and NC controls. For voxel-wise analyses, artifacts (edges and capillaries) were masked in the background-subtracted 6E10-immunofluorescence images, and voxels not associated with reactive microglia were masked the background-subtracted iba1-immunofluorescence images. The resulting images were then warped to atlas space, z-scored, and smoothed for intensity-based voxel-wise comparisons, followed by false discovery rate correction (q < 0.2). Clusters of significant voxels were warped back to full-resolution tissue space to measure label densities (the volume of segmented 6E10 aggregates / cluster volume * 100 or the volume of segmented reactive microglia / cluster volume * 100). Clusters were considered valid if unpaired one-tailed t-tests confirmed differences between the groups. (B, C) Valid clusters are shown in 3D brain models with Allen brain atlas coloring. For display purposes, unilateral cluster maps were mirrored. (D and F) The regional composition of valid clusters by volume is shown in sunburst plots (outer rings represent subregions, whereas inner rings correspond to parent regions). (E and G) Bar graphs summarize data for all valid clusters. n = 4 for all groups, except for 5XFAD iba1 where n = 3 (one sample was excluded due to a technical error in sample preparation). Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001. Supplemental Table S9 defines region abbreviations, and Supplemental Table S10 reports the significance, volumes, positions, and regional composition of valid clusters. Numbers ([‘2/3’, ‘5’, ‘6a’]) = cortical layers. Additional abbreviations not defined in Supplemental Table S9 include: {‘c’: ‘central nucleus’, ‘d’: ‘dorsal nucleus or dorsal’, ‘e’: ‘external nucleus’, ‘me’: ‘median’, ‘mot’: ‘motor related’, ‘sen’: ‘sensory related’, ‘v’: ‘ventral’}