Ova fluorescein conjugate

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.

CHI (M_w = 190,000‒310,000 g/mol), ALG (M_w = 120,000‒190,000 g/mol), TA, DOX, and FITC were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mg⁺ and Ca²⁺-free 10× PBS was purchased from Gibco (Waltham, MA, USA). The hyaluronic acid was purchased from SK Bioland (Cheonan, Chungnam, South Korea). The Ova was purchased from Bio Basic Inc. (Konrad Crescent, Markham, Ontario, Canada). The Ova fluorescein conjugate was purchased from Invitrogen (Carlsbad, CA, USA).