Aberrant distribution of exosomes in lymphoid tissues of CD169^−/−in vivo. C57BL/6 or CD169^−/− mice were (A [spleen],B,C,F) intravenously (IV) or (A [LN],E) subcutaneously (SC) injected in the forelimb with 100 or 50 µg of biotinylated B cell–derived exosomes (Exo-bio; purified by ultracentrifugation), respectively. (D,F,G) Alternatively, C57BL/6 or CD169^−/− mice were IV or SC injected with 2 × 10¹¹ or 1 × 10¹¹ 100 nm fluorescent microspheres, respectively (green). For IV or SC routes, mice were killed at 5 minutes with spleens and livers harvested or at 30 min and draining LN harvested, respectively. (A-C,E) Exo-bio was detected with streptavidin-Alexa-594 (red). Sections were colabeled for (A,G) marginal metallophilic or subcapsular sinus macrophages with anti-CD169 (MOMA-1), (B) MZ or red pulp macrophages with anti–SIGN-R1 (ER-TR9) or anti-F4/80, respectively, or (C) Kupffer cells with anti-F4/80. Primary antibodies were detected with (A-C) anti-rat IgG-Alexa-488 (green) or (G) anti-rat IgG-Alexa-594 (red), and nuclei were counterstained with DAPI (blue). Original magnification, (A [LN],D[spleen],E) ×100 , (A [spleen],B,D [LN],G) ×200, and (C) ×400. Bar represents (A [spleen],B,D [LN],G) 200 µm, (A [LN],D [spleen],E) 250 µm, and (C) 50 µm. All results are representative of ≤4 mice per group. (F) Percent colocalization was calculated from fluorescent microscopy photos of (A [spleen],B,G) spleen sections. Ten individual photographs per mouse (original magnification, ×200) were analyzed for colocalization of green (Alexa-488) and red (Alexa-594) signal using the Manders’ coefficient with ImageJ. Each point represents an individual photograph; line indicates mean. Circles, exosomes; squares, beads; closed symbols, C57BL/6 mice; open symbols, CD169^−/− mice. One-way ANOVA with Bonferroni postcorrection test was performed: ns, not significant; *P < .05; **P < .01; ****P < .0001.