Goat anti mouse igg conjugated to alexa555

After being treated as indicated previously, cellular distribution of ZO-1 and Occludin was visualized by immunofluorescence as previously reported [14 (link), 21 (link)]. In brief, after being rinsed with PBS, filters were fixed in 100% methanol overnight at −20°C and 100% acetone at −20°C for 1 min. Subsequently, at room temperature, filters were blocked with 1% BSA for 2 h and then treated with anti-mouse Occludin (4 μg/ml) and anti-rabbit ZO-1 (6 μg/ml) at 4°C overnight. After being washing with PBS, filters were incubated with goat anti-mouse IgG conjugated to Alexa555 (Molecular Probes, Eugene, OR, USA) and goat anti-rabbit IgG conjugated to Alexa488 (Molecular Probes, Eugene, OR, USA) in 1% BSA at room temperature for 1 h and then rinsed with PBS. In the next step, the Prolong Gold Antifade Reagent (Molecular Probes, Eugene, OR, USA) was used, and cells were stored at 4°C in dark until analysis. Under Fluoview 1000 confocal microscope (Olympus, Tokyo, Japan), the fluorescence was visualized.

Caco-2 monolayers were treated as indicated above; then, the localization of TJ protein ZO-1, Occludin, and NF-κB p65 in cells was evaluated by immunofluorescence as described previously [26 (link)]. In short, filters were washed with PBS and fixed in 100% methanol at -20°C for the night, then fixed by 100% acetone at -20°C for 1 min. Next, filters were washed with PBS and blocked with 1% BSA at room temperature for 2 h. Then, the filters were incubated with anti-rabbit ZO-1 (1 : 100 dilution, Invitrogen, USA) and anti-mouse Occludin (1 : 200 dilution, Invitrogen, USA) or anti-rabbit NF-κB p65 (1 : 100 dilution, CST, USA) antibody at 4°C for the night. After PBS washing, goat anti-rabbit IgG conjugated to Alexa488 (Molecular Probes, USA) and goat anti-mouse IgG conjugated to Alexa555 (Molecular Probes, USA) were used to incubate with filters in 1% BSA at room temperature for 1 h. Then, the cells were washed with PBS and subsequently stained with sodium iodide. After PBS washing, the Prolong Gold anti-fade reagent (Molecular Probes, USA) was used to mount cells; then, the cells were stored at 4°C in the dark until analyzed. The fluorescence was visualized under a Fluoview 1000 confocal microscope (Olympus, Japan).