Nanoparticle-Ovalbumin Immunization Protocol

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.