Actin binding phalloidin alexa350

For these studies six to eight week-old female C57BL/6 mice (Jackson Laboratories) were used. Chicken Ova (Sigma) was used as a model protein antigen. Carboxylate-modified fluorescent polystyrene nanoparticles (20 nm and 40 nm, Invitrogen), were used as a model particulate antigen, as well as Ova antigen carriers for immunizations. For immunization experiments 20 nm NPs were conjugated to Ova and every batch of conjugated NPs was analyzed by dot-blot as described previously [17] (link). Biotinylated rabbit anti-Ova antibodies (Thermo Scientific) in combination with streptavidin-FITC (eBioscience) were used to detect Ova antigen and NP-Ova. To highlight the tissue architecture in tissue cryosections, actin-binding Phalloidin-Alexa350 (Invitrogen) was used. Tissue staining with biotinylated Lyve-1 (eBioscience) and E-cadherin (BD Biosciences) antibodies, followed by FITC-conjugated streptavidin (eBioscience) was done in order to visualize the lymphatics and the FRT epithelium. Some tissue sections were stained with antibodies specific for CD11c+ DCs (eBioscience). All antibodies were used at a 1∶100 dilution in blocking buffer.

To confirm the presence of i.n.-applied antigens within the respiratory tract, 20 nm NPs (10% from an original concentration of 2%) and soluble antigens (Ova (5 mg/20 µL, Sigma), lysine-fixable biotinylated dextran (10 K MW, Invitrogen)) were i.n. administered to anesthetized mice in a volume of 20 μL (10 μL/ nostril). At 1 h after antigen administration mice were euthanized, lung tissues were excised and snap-frozen in optimum cutting temperature (O.C.T.) freezing medium. Tissue cryosections were fixed in 10% PFA then stained with streptavidin-FITC (fluorescein isothiocyanate) and antibodies specific for Ova and DC marker CD11c (eBioscience). Tissue architecture was highlighted with actin-binding phalloidin-Alexa350 (Invitrogen). Stained tissue sections were imaged with a Leica DM1000B fluorescence microscope and acquired images were analyzed as described previously [34 (link),38 (link),39 (link)]. To get a more detailed view of the tissue, lung sections were also stained with hematoxylin and eosin (H&E) and imaged with an Olympus BX41 microscope equipped with an Olympus DP72 camera using CellSens Standard imaging software.