Extended Data Figure 3. Assessment of Vcam1fl/flSlco1c1-CreERT2+/− young mice and Sudan Black B treatment quenches autofluorescent staining caused by lipofuscin revealing VCAM1 cerebrovascular specificity, and immunodeficient mice exposed to aged human plasma over 3 weeks have increased brain aging hallmarks.

(a) Schematic. n= 5 mice/group.

(b) Quantification in the DG of VCAM1 from immunostained confocal images. n= 5 mice/group. Unpaired two-tailed Student’s t-test. Mean +/− SEM. *p=0.0451.

(c) Quantification in the DG of BrdU+ and Sox2+ NPCs and triple labeled GFAP+ neural stem cells from confocal images of immunostained sections. Scale bar = 100 μm. n= 5 mice/group. Unpaired two-tailed Student’s t-test. Mean +/− SEM. ***p=0.007, **p=0.0227, *p=0.0038.

(d) Quantification in the DG of Iba1 and CD68 from confocal images of immunostained sections. n= 5 mice/group. Unpaired two-tailed Student’s t-test. Mean +/− SEM. *p= 0.0454.

(e) Experimental Design. n= 6 Cre− and 7 Cre+ mice per group.

(f) Quantification of VCAM1+ percent area in lectin+ vasculature of immunostained sections from 6 Cre− and 5 Cre+ mice/group. ****p<0.0001. Unpaired two-tailed Student’s t-test. Mean +/− SEM.

(g) Quantification of the total number of BrdU+ cells, BrdU+Sox2+ co-labeled neural progenitor cells, and (h) average # DCX+ imature neurons per section in the DG of immunostained sections. n=6 Cre− and 7 Cre+ mice per group. *p=0.0012, **p=0021, ***p=0.0028. Unpaired two-tailed Student’s t-test. Mean +/− SEM.

(i) Quantification of Iba1 and CD68 in the DG of immunostained sections. n=6 Cre− and 7 Cre+ mice per group. Bars represent mean. Error bar represents SEM. Stain experiment repeated twice with similar results; Similar mouse experiments using these validated transgenic mice repeated 4 times with similar results (see Supplementary Table 4).

(j) Confocal images of brain sections of Cre+ or Cre− aged Slco1c1-CreERT2-Vcam1fl/fl mice treated with tamoxifen in young adulthood (age 2 months) and aged to 18 months stained for anti-VCAM1 or IgG isotype control, Aqp4, and GFAP. Hoechst labels cell nuclei. Aged (18-month-old) brain sections were treated with Sudan Black B to remove lipofuscin background in the granular and hilus layers of the DG. SBB treatment removes the majority of lipid-based artifacts typically seen in aged tissues without suppressing immunofluorescent labeling. Scale bar = 100 μm. Experiment repeated three times with similar results.

(k) Aged (18-month-old) Cre+ and Cre− brain sections were immunostained using the regular protocol, without Sudan Black B treatment. Heavy lipofuscin background is present in the Cy3 fluorescence channel. Experiment repeated three times with similar results.