Fig. 5

Dual staining with BSB and MCAAD-3 detected subtle pathology in the greater non-plaque parenchyma of 5xFAD mouse brains. (A, D) Representative spectral micrographs of gray matter sections from WT and 5xFAD mice. (B, E) The greater parenchyma (all plaques omitted) was analyzed with spectral phasor. In contrast to plaques (Figs. 2 and 4), the distributions did not show obvious differences. Instead, the ROI (pink rectangle) shown on the phasor surface was determined using phasor optimization to find the population of kernels which were most significantly different between the WT and 5xFAD groups. These kernels are shown in blue in C and F, comprising ≈ 85% of the background area, suggesting diffuse and widespread subtle deposition of amyloid in the 5xFAD samples. (G) Based on phasor analysis, a significant parenchymal difference was observed between WT and 5xFAD based on the calculated r*θ values (Wilcoxon signed-rank test, p = 0.04). Notably, the r*θ values of the 5xFAD background moved in the direction of the amyloid plaques. The difference between the backgrounds was abolished using formic acid (FA) treatment (r*θ values calculated for the same ROIs of the samples that were treated with formic acid and restained shown in H), supporting the notion that the differences in G were driven by subtle amyloid deposition. Scale bars: 50 μm, error bars indicate SEM