Dextran fluorescein

WT and cKO BMDCs were incubated with dextran–fluorescein (10,000 MW; Invitrogen) at a concentration of 1 μg/ml for 30 and 60 min at 37°C, washed three times with PBS, and then fixed with 4% PFA. Next, the cells were stained with anti-EEA1 and DAPI, before being analyzed as described above in immunofluorescence imaging analyses. Uptake of dextran–fluorescein was quantified using ImageJ software. The total intensity of the dextran–fluorescein taken up by cells was measured and was divided by the number of cells in each photograph to obtain average dextran–fluorescein taken up per cell (∼40 cells were analyzed per time point for each genotype).

Agarose (Fischer Scientific, BP1356–100), Bovine Serum Albumin (Sigma, A4503), Fibronectin (Sigma, F1141), dextran fluorescein (Invitrogen, D1820), dextran tetramethylrhodamine (Molecular Probes life technologies, D3312), Gelatin (Sigma, G1890), Methylcellulose (Sigma, M0387–100), Mowiol 40–88 (Fluka, 81386), Penicillin-Streptomycin (Sigma, P4458), Sucrose (VWR, 27480.294). Danilchick’s medium 1 × (DFA): NaCl (53 mM), NA2CO3 (5 mM), K Gluconate (4.5 mM), Na Gluconate (32 mM), MgSO4–7H20 (1 mM), CaCl2 (1 mM), BSA 0.1%. MEMFA: MOPS (1 mM), EGTA (2 mM), MgSO4 (1 mM), Formaldehyde (3.7%). Normal Amphibian Medium (NAM): NaCl (110 mM), KCl, (2 mM), Ca(CO3)2 (1 mM), MgSO4 (1 mM), EDTA (0.1 mM), NaHCO3 (1 mM), Sodium Phosphate (2 mM).NTMT: NaCl (0.1 M), TrisHCl pH9.5 (0.1 M), MgCl2 (50 mM), Tween 0.1%. Phosphate Buffer Saline 1 × : NaCl (137 mM), KCl (2.7 mM), Na2HPO4 (10 mM), KH2PO4 (1.8 mM), CaCl2–2H2O (1 mM), MgCl2–6H2O (0.5 mM). Human Sdf1 (Calbiochem, 572300; used at 0.5 μg/mL in solution). Mouse Semaphorin-3A-Fc (R&D Systems, 5926-S3; used at 15, 30 or 60 ng/mL coated or added in solution), mouse Semaphorin-3F-Fc (R&D Systems, 3237-S3; used at 120, 240 or 480 ng/mL coated).

Six to ten week-old C57BL/6 mice (Jackson laboratories) were used for the studies. Carboxylate-modified fluorescent polystyrene NPs, ranging in size from 20 nm to 2 µm (Invitrogen), and E.coli BioParticles® (Invitrogen) were used as model particulate antigens. Chicken Ova (45 kDa, Sigma), Ova-fluorescein conjugate (Invitrogen), dextran-fluorescein, lysine-fixable dextran-biotin (40 kDa, Invitrogen), and LPS-Alexa Fluor® 488 (3 kDa, Invitrogen) were used as model soluble antigens. Biotinylated rabbit anti-Ova antibodies (Thermo Scientific) and streptavidin-FITC (Biolegend) were used to detect Ova and Ova conjugated to NPs (NP-Ova). Anti-CD11c (eBioscience), Cy-18 (Biolegend) and Lyve-1 (eBioscience) antibodies were used to label LP DCs, goblet cells, and lymphatic ducts respectively. A combination of monoclonal mouse anti-E-cadherin (BD Biosciences) primary antibody and goat anti-mouse-FITC (BD Biosciences) secondary antibody was used to label the IECs. All antibodies were used at a 1∶100 dilution in appropriate blocking buffer. To highlight the tissue architecture in cryosections, actin-binding Phalloidin-Alexa 350 (Invitrogen) was used. DAPI (4′,6-Diamidino-2-Phenylindole, Dilactate, Invitrogen) was used for in vivo labeling of the IEC nuclei. Genistein and chlorpromazine (CPZ) (Sigma) were used for in vivo inhibition of NP uptake at 200–1000 µM and 10–100 µg/ml respectively.

Migrating cells from the afferent lymph were cultured [IMDM containing 10 % FCS and 10⁻⁵M 2-β-mercaptoethanol (Sigma-Aldrich)] with the recombinant viruses using optimal MOI values, as described previously (Cubillos-Zapata et al., 2011 (link)). In some assays, transferrin-AF568 (Sigma) and dextran-fluorescein (Invitrogen) were used as tracing markers. In neutralization assays, AdV5 was incubated with 1 µg of anti-hexon antibody raised in goats (Millipore) or with bovine pre-immune or hyperimmune sera raised against human AdV5 generated by immunizing calves with 1×10⁹ virus particles of AdV5 as described above.

In tail lymphatic vessel lymphangiography, 5 mg/ml 2000 kDa Dextran-Rhodamine (D7139, Invitrogen) was injected directly into the tail lymphatics using an oil injector under fluorescence stereomicroscopy. The video was taken immediately after injection. For lymphangiography in the trunk, 1-2 nl 10 mg/ml 2000 kDa Dextran-Rhodamine was directly injected into the trunk. After 10-30 min, the larvae were embedded for confocal imaging. Adult retro-orbital injection was performed as described before⁹⁸ with modifications. Injections could be made into the tissue behind the eyes as well as the retro-orbital venous sinus. The surrounding lymphatic vessels could take up the Dextran-fluorescein and finally entered the blood circulation. A 10 μl hamilton syringe was used, and the tip was hand honed to be shorter and smoother. Adult fish was anesthetized with 0.2 mg/ml tricaine and positioned dorsal up, head right. The needle was inserted 1-2 mm deep at a 45-degree angle to the fish at seven o’clock position. 3 μl 5 mg/ml 2000 kDa Dextran-fluorescein (D7137, Invitrogen) was injected. For the angiography at 4 dpf, 1-2 nl 5 mg/ml 2000 kDa Dextran-Rhodamine was injected into the CCV. Dextran-Rhodamine would be observed circulating in the bloodstream immediately after injection. The successful injection ratio was around 50%.