Mouse anti claudin 4

When Caco-2 cells had been incubated with the bacteria as described above without FITC-dextran for 11 h at +37°C in 5% CO₂, the cells were washed four times with PBS, and then fixed with 200 μl/insert of -20°C methanol for 5 min, followed by an additional four washes with PBS. One drop/insert of Image-iT FX signal enhancer (Life Technologies) was added and the cells were incubated for 30 min at room temperature to block the unspecific binding sites. After washing with PBS, the cells were incubated with 100 μl/insert of rabbit anti-occludin (1:500; Invitrogen), rabbit anti-ZO-1 (1:100; Thermo Scientific), or mouse anti-claudin-4 (1:1000; Invitrogen) antibodies diluted in PBS at room temperature for 1 h, washed four times with PBS, and then incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:1000; Invitrogen) or Alexa Fluor 594 goat anti-mouse IgG (1:1000; Invitrogen) diluted in PBS at room temperature for 1 h. After washing four times with PBS, the Thincert membranes were cut off and mounted on glass slides in Fluoprep medium (BioMérieux). The cells were observed for tight junction proteins using fluorescence microscopy (Leica DM400B).