Tritc conjugated goat anti rabbit igg

The paraffin sections were incubated in the rabbit polyclonal antibody β3-AR (1∶50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. As a negative control, PBS was used in place of the primary antibody. Then TRITC-conjugated goat anti-rabbit IgG (1∶100 dilution, Jackson ImmunoResearch, West Grove, PA, USA) was used as the secondary antibody to detect the primary antigen-antibody reaction. The nuclei was stained by 4′-6-diamidino-2-phenylindole (DAPI) dye (1∶2000 dilution, Sigma, St Louis, MO, USA). Immunofluorescent labeling of the sections were observed with a fluorescence microscope (Nikon Eclipse 55i, Nikon, Tokyo, Japan). Quantification of the β3-AR fluorescence density was determined by IPP 6.0.

Caco-2/TC7 cells differentiated on Transwell filters (1×10⁶ cells/filter), were untreated (control) or apically treated with 1 mL of medium containing ETEC (5×10⁶ CFU/mL) or L. amylovorus (5×10⁷ CFU/mL), either alone or simultaneously, for 2.5 hrs. At the end of treatment, cells were washed with PBS and fixed in ice-cold methanol for 3 min. Localizations of TLR4, P-p65, and occludin were analyzed as follows. Briefly, the cells were treated with rabbit polyclonal anti-TLR4 or P-p65 antibodies (Cell Signaling Technology), or mouse monoclonal anti-occludin antibody (Zymed Laboratories, Milan). For secondary detection, the cells were incubated with FITC-conjugated goat anti-rabbit IgG for TLR4, TRITC-conjugated goat anti-mouse IgG (Jackson Immunoresearch, Milan) for occludin or TRITC-conjugated goat anti-rabbit IgG for P-p65. For P-p65 analysis, the cell nuclei were stained with 300 nM DAPI, added directly to the mounting medium. Stained monolayers were mounted on glass slides by using Prolong Gold antifade Reagent (Molecular Probes, Invitrogen, Milan) and analyzed under a confocal microscope (LSM 700, Zeiss, Jena, Germany).

Anti-RelB (CAT# 10544), anti-p52 (CAT# 37359), and rabbit anti-Flag (CAT# H2368) antibodies were purchased from Cell Signaling Technology. Mouse anti-Myc (CAT# 05-724), anti-p24 (CAT# MAB880-A), anti-RNA Pol II (CAT# 05-623B), normal mouse IgG (CAT# 12-371B) and normal rabbit IgG (CAT# 12-370) were from Millipore. Mouse anti-Flag (M2) (CAT# F1804), mouse anti-HA (CAT# H3663), and rabbit anti-EGFP (CAT# G1544) antibodies were obtained from Sigma. Anti-GAPDH (CAT# sc-32233), anti-Tubulin (CAT# sc-32293), rabbit anti-HA (CAT# sc-805), rabbit anti-Myc (CAT# sc-789), mouse anti-EGFP (CAT# sc-9996) and horseradish peroxidase-conjugated secondary antibodies (CAT# sc-2005 and sc-2004) were from Santa Cruz Biotechnology. FITC-conjugated goat anti-mouse IgG (CAT# 115-095-005) and TRITC-conjugated goat anti-rabbit IgG (CAT# 111-025-003) were from Jackson Immuno Research. RNase A (CAT# 20-297) was purchased from Millipore. DAPI (4′,6-diamidino-2-phenylindole) (CAT# D8417) and Phorbol 12-myristate 13-acetate (PMA) (CAT# P8139) were purchased from Sigma.