Mouse monoclonal anti e cadherin

The samples were fixed in 4% paraformaldehyde and processed as per the standard procedure. Six-micron-thick sections were prepared for hematoxylin/eosin (H&E) staining and immunostaining. The specimens were boiled in citrate buffer (pH 6.0) for antigen retrieval and blocked using 1% goat serum or 5% bovine serum albumin in PBS. The specimens were incubated with primary antibodies against monoclonal rabbit anti-Ahnak (1:200, produced by Prof. Bae’s lab, Department of Life Sciences, Ewha Woman’s University, Seoul, Korea), monoclonal rabbit anti-p63 (1:200, GeneTex), monoclonal rabbit anti-upk1b (1:200, Invitrogen), monoclonal mouse anti-E-cadherin (1:200, BD Transduction), monoclonal rabbit anti-c-kit (1:100, Abcam), monoclonal mouse anti-α-SMA (1:200, Invitrogen) at 4 °C overnight. The following day, these sections were incubated with a secondary antibody Alexa488-conjugated goat-anti-rabbit IgG (1:200, Invitrogen), Alexa488-conjugated goat-anti-mouse IgG (1:200, Invitrogen), Alexa555-conjugated goat-anti-mouse IgG (1:200, Invitrogen), Alexa555-conjugated goat-anti-rabbit IgG (1:200, Invitrogen) and counterstained with TO-PROTM-3 (T3605, Invitrogen, OR, USA; 1:1000) to visualize nuclei. All specimens were examined using a confocal laser microscope (DMi8, Leica, Germany).

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.