Biotinylated rabbit anti ova antibodies

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 that the NPs were conjugated to Ova, NPs, Ova, or Ova-conjugated NPs (NP-Ova) were spotted on a 0.2 µm nylon membrane. The membrane was washed with Tris-buffered solution containing 0.1% Tween 20 (Sigma) (TTBS), blocked with TTBS containing 5% skim milk (Difco) for 1 hour then incubated with biotinylated rabbit anti-Ova antibodies (Thermo Scientific) for 1 hour. After 3 washes the membrane was incubated with streptavidin-FITC (Biolegend) for 1 hour, washed, dried then imaged with a Leica DM4000B fluorescent microscope using a 2.5× objective.

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.