*2.1. Immunohistochemistry*

ABCG2 was immunohistochemically stained in brain slices of APP/PS1-21 mice and wild-type littermates aged 6 months (*n* = 3 per group) (Figure 1). For immunohistochemical analysis, we selected two brain regions in which Aβ load was shown to be high in APP/PS1-21 mice at the investigated age (hippocampus and cortex) and one region with negligible Aβload (cerebellum) [33]. A semi-quantitative analysis of the stained microvessels indicated a significant decrease (by 29–37%) in the number of ABCG2-stained microvessels in the hippocampus and cerebellum of APP/PS1-21 versus wild-type mice (Figure 2a,c), while no significant difference was found in the cortex (Figure 2b). This suggested that the reduction in the abundance of ABCG2 in APP/PS1-21 mice was independent of Aβ deposition.

**Figure 1.** Examples of immunohistochemical staining of mouse ABCG2 in microvessels of 6-months-old wild type and APP/PS1-21 mice in (**a**) hippocampus (dentate gyrus), (**b**) cortex (cingulate cortex) and (**c**) cerebellum (4th and 5th cerebellar lobules with primary fissure) region. Enlarged areas are shown at 20× magnification. Scale bar in lower left indicates 50 µm. (NC, negative control: staining protocol without primary antibody).

**Figure 2.** Semi-quantitative analysis of ABCG2-stained microvessels in (**a**) hippocampus, (**b**) cortex and (**c**) cerebellum of 6-months-old APP/PS1-21 mice and age-matched wild-type mice. For each region, the mean of four visual fields (at 20× digital magnification) per animal (*n* = 3 animals per strain) was used for statistical testing. Error bars indicate SD. (ns, not significant; \*\* *p* < 0.01; \*\*\* *p* < 0.001; 2-sided *t*-test).
