(A) Overview of the zebrafish head region depicting intratectal injection of fluorescent dyes into the center of the TeO close to the meninges in 5dpf embryos. Red inset denotes area of image detail for representative dorsal confocal projections in B–I. (B) and (C) In DMSO treated fish co-injection of pHr and IgG-647 reveals high degree co-localization of both tracers in vesicles within mCitrine positive BLECs (B, white arrowheads). Inhibition of dynamin-dependent endocytosis with Pyrimidyn-7 results in reduced signal intensity from IgG-647 positive vesicles and an almost complete block of pHr uptake (C, white arrowheads). (D–F) Separate, consecutive injections of BSA or mannan with pHr and IgG-647. Injection of BSA, pHr and IgG-647 results in co-localization of both tracers in BLECs (D, white arrowheads). In contrast, initial injection of mannan completely blocks the uptake of pHr but not the accumulation of IgG-647 to BLECs (E, white arrowheads). However, initial injection of pHr followed by mannan and IgG-647 results in accumulation of both tracers in BLECs with hardly any co-localization (F, white arrowheads and arrows). (G–I) Same experimental setup as in D–F except the embryos were fixed in PFA five minutes after the last injection to enable higher resolution imaging. Area within the white dotted lines in 3D-reconstructions reveals the lumen of BLECs (G’,H’ and I’). In BSA control injected fish both dyes co-localize in intraluminal vesicles (G and G’, white arrowheads). In mannan injected fish, IgG-647 cannot be internalized completely but remains stuck at the cell surface, (H and H’, white arrowheads). Finally, initial injection of pHr reveals internalized vesicular pHr and membrane associated IgG-647 (I and I’, white arrows and white arrowheads). Data are representative of at least two independent experiments. BLEC, brain lymphatic endothelial cell; BSA, bovine serum albumin; dpf, days post fertilization; IgG-647, IgG-conjugated Alexa Fluor 674; PFA, paraformaldehyde; pHr, pHrodo Red Avidin; TeO, Optic Tectum.