Extended Data Fig. 3|. BBB permeability and homeostasis after plasma injections, and brain uptake of plasma across injection volumes and chemistries.

a, Assessment of perfusion completeness by measuring residual, intravenously injected 2 mDa dextran-FITC (DFITC) in brain tissue. No significant difference between dextran-FITC-injected and PBS-injected young (3-month-old) mice after perfusion (n = 6 PBS unperfused and n = 8 for the others; ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons correction; mean ± s.e.m.). b, Circulating 2 mDa dextran-FITC in plasma at the time of death in d (n = 6 PBS unperfused and n = 8 for the others; ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons correction; mean ± s.e.m.). c, d, BBB permeability after exposure to 150 μl saline or plasma was probed the next day by injections of 3-kDa (c) and 70-kDa (d) dextran tracers for quantification by fluorescence plate reader. Mild traumatic brain injury (TBI) served as a positive control for BBB leakage (for 3 kDa dextran: n = 8 saline, n = 9 plasma, n = 7 TBI; for 70 kDa dextran: n = 4 saline and TBI, n = 5 plasma; one-way ANOVA with Tukey’s multiple comparisons correction; mean ± s.e.m.). e, Representative images of endogenous immunoglobulin (white) extravasation into the parenchyma after exposure to 150 μl saline or plasma, or TBI⁷⁵. Scale bars, 50 μm. f, Quantification of endogenous immunoglobulin (IgG) extravasation into the brain parenchyma after exposure to 150 μl saline or plasma, or TBI (n = 4 saline, n = 6 plasma and TBI; ns P = 0.9995, one-way ANOVA with Tukey’s multiple comparisons correction; mean ± s.e.m.). g, Plasma protein concentration of mice injected with 150 μl of plasma, 20 h later at time of death in young (3-month-old) and aged (20–24-month-old) mice used throughout the study compared to plasma concentrations at baseline (n = 35 young baseline, n = 35 young injected, n = 22 aged baseline, n = 32 aged injected; ns (left to right) P = 0.9923, P = 0.0657, P = 0.0657, P = 0.4302, P = 0.5430, two-way ANOVA with Sidak’s multiple comparisons test; mean ± s.e.m.). h, t-SNE plot of brain endothelial cells from young (3-month-old) mice at baseline and after exposure to 150 μl of plasma, demonstrating no significant perturbation of the cerebrovascular transcriptome upon plasma transfer (n = 3 mice). i, Representative brain images of Atto 647N-labelled plasma injected at various volumes, assayed 4 h later. 150 μl corresponds to 10–15 mg (0.5 mg/g body weight). CD31 marks brain endothelial cells. Scale bars, 100 μm. j, Representative brain images after Atto 647N-labelled plasma transfusion and stained for mouse albumin (mAlbumin; top) and mIgG (bottom). Minority of capillaries are albumin and IgG⁺ capillaries (arrowheads). Scale bars, 40 μm (left) and 10 μm (right). k, Atto 647N-labelled plasma (left), Alexa Fluor 647-labelled plasma (middle) and l-azidohomoalanine-labelled plasma, detected by sDIBO-647 click strain-promoted alkyne-azide cycloadditions (SPAAC) slice staining. In vivo labelled l-azidohomoalanine plasma^76,77 was extracted and transfused into separate mice under normal (no l-azidohomoalanine) conditions. Scale bars, 20 μm. l, Representative images of after Atto 647N-labelled plasma transfusion and stained for mouse transferrin (top). Scale bars, 50 μm. In addition, quantification of the per cent of plasma⁺ neurons that also contain transferrin is shown (bottom, n = 4).