Figure S2.
Additional analysis of tracer uptake, ultrastructure, and caveolar transcytosis in DRG endothelium. (A) Low-magnification images of Iba1 and CD31 stained DRG sections from mice injected with indicated i.v tracers. All tracers readily accumulate inside macrophages. Images related to analyses in Fig. 3 A and panel B. Scale bar, 50 μm. (B) Uptake of indicated i.v injected tracers in DRG endothelial cells, separated by vessel segments. The following tracers, doses, and circulation times were used: 3 kD dextran-TMR (25 mg/kg, 1 h, n = 4 mice), BSA-A647 (5 mg/kg, 1 h, n = 4 mice), 70 kD dextran-TMR (25 mg/kg, 1 h, n = 9 mice), goat anti rabbit IgG-A488 (4 mg/kg, 4 h, n = 4 mice), 500 kD dextran-FITC (25 mg/kg, 1 h or 24 h, n = 4 mice), 2,000 kD dextran-FITC (25 mg/kg, 24 h, n = 4 mice). Values are mean of individual macrophages, normalized to tissue background. (C) Analysis of endothelial fenestrae (arrowheads) in TEM images from indicated DRG vessel segments. Total images per vessel segment are indicated above each bar. n = 4 mice. Fisher’s exact test. ***P < 0.001. Scale bar, 1 μm. (D) Scanning electron micrograph of luminal surface of DRG v-cap with an apparent scarcity of endothelial fenestrae. Scale bar, 500 nm. (E) Representative TEM images of v-caps from WT and Cav1−/− mice, showing the absence of caveolar vesicles (red arrowhead), but presence of fenestrae (black arrowhead) in Cav1−/− mice. Scale bar, 500 nm. (F)Mfsd2a expression across endothelial cells isolated from indicated organs showing expression restricted to brain and testis (Kalucka et al., 2020). (G)Mfsd2a expression in MACS-enriched CD31+ endothelial cells from indicated organs, normalized to brain. n = 3 (brain), 4 (DRG), 2 (kidney, liver, lung). (H) MFSD2A immunostaining in DRG section, demonstrating that MFSD2A is absent in SpN and DR. Scale bar, 200 μm. (I) CAV1 immunostaining in entire DRG section from WT and Cav1−/− mice, demonstrating antibody specificity. Scale bar, 200 μm. (J) Representative MFSD2A immunostaining in DRG section, visualizing cropped areas in Fig. 3 D. Scale bar, 100 μm. (K) Representative CAV1 immunostaining in DRG section, visualizing cropped areas in Fig. 3 D. Scale bar, 100 μm. A, artery; V, vein.

Additional analysis of tracer uptake, ultrastructure, and caveolar transcytosis in DRG endothelium. (A) Low-magnification images of Iba1 and CD31 stained DRG sections from mice injected with indicated i.v tracers. All tracers readily accumulate inside macrophages. Images related to analyses in Fig. 3 A and panel B. Scale bar, 50 μm. (B) Uptake of indicated i.v injected tracers in DRG endothelial cells, separated by vessel segments. The following tracers, doses, and circulation times were used: 3 kD dextran-TMR (25 mg/kg, 1 h, n = 4 mice), BSA-A647 (5 mg/kg, 1 h, n = 4 mice), 70 kD dextran-TMR (25 mg/kg, 1 h, n = 9 mice), goat anti rabbit IgG-A488 (4 mg/kg, 4 h, n = 4 mice), 500 kD dextran-FITC (25 mg/kg, 1 h or 24 h, n = 4 mice), 2,000 kD dextran-FITC (25 mg/kg, 24 h, n = 4 mice). Values are mean of individual macrophages, normalized to tissue background. (C) Analysis of endothelial fenestrae (arrowheads) in TEM images from indicated DRG vessel segments. Total images per vessel segment are indicated above each bar. n = 4 mice. Fisher’s exact test. ***P < 0.001. Scale bar, 1 μm. (D) Scanning electron micrograph of luminal surface of DRG v-cap with an apparent scarcity of endothelial fenestrae. Scale bar, 500 nm. (E) Representative TEM images of v-caps from WT and Cav1−/− mice, showing the absence of caveolar vesicles (red arrowhead), but presence of fenestrae (black arrowhead) in Cav1−/− mice. Scale bar, 500 nm. (F)Mfsd2a expression across endothelial cells isolated from indicated organs showing expression restricted to brain and testis (Kalucka et al., 2020). (G)Mfsd2a expression in MACS-enriched CD31+ endothelial cells from indicated organs, normalized to brain. n = 3 (brain), 4 (DRG), 2 (kidney, liver, lung). (H) MFSD2A immunostaining in DRG section, demonstrating that MFSD2A is absent in SpN and DR. Scale bar, 200 μm. (I) CAV1 immunostaining in entire DRG section from WT and Cav1−/− mice, demonstrating antibody specificity. Scale bar, 200 μm. (J) Representative MFSD2A immunostaining in DRG section, visualizing cropped areas in Fig. 3 D. Scale bar, 100 μm. (K) Representative CAV1 immunostaining in DRG section, visualizing cropped areas in Fig. 3 D. Scale bar, 100 μm. A, artery; V, vein.

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