Tetramethylrhodamine isothiocyanate tritc conjugated secondary antibodies

Caco-2/TC7 cells were washed three times with cold PBS containing Ca⁺⁺ and Mg⁺⁺, fixed in ice-cold methanol for 3 min, and then incubated with rabbit polyclonal anti-ZO-1, mouse monoclonal anti-occludin, mouse monoclonal anti-β-catenin, or rabbit polyclonal anti-E-cadherin antibodies (Zymed Laboratories), for 1 h. For secondary detection, the cells were incubated with fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) conjugated secondary antibodies (Jackson Immunoresearch, Milan, Italy), for 1 h. Stained monolayers were mounted on glass slides by using Prolong Gold antifade Reagent (Molecular Probes, Invitrogen, Milan, Italy) and analyzed using a fluorescence microscope (Zeiss, Jena, Germany).

The protective effects of LS on membrane damages induced by oxidative stress were assessed evaluating tight and adherent junctions’ (AJs) principal proteins immunolocalization. Briefly, Caco-2/TC7 cells were washed three times with cold PBS containing Ca⁺⁺ and Mg⁺⁺, fixed in ice-cold methanol for 3 min, and then incubated with rabbit polyclonal anti-ZO-1, mouse monoclonal anti-occludin, mouse monoclonal anti-β-catenin, or rabbit polyclonal anti-E-cadherin antibodies (Zymed Laboratories), for 1 h. For secondary detection, the cells were incubated with fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) conjugated secondary antibodies (Jackson Immunoresearch, Milan, Italy), for 1 h. Stained monolayers were mounted on glass slides using the Prolong Gold antifade reagent (Molecular Probes, Invitrogen, Milan, Italy) and analyzed using a fluorescence microscope (Zeiss, Jena, Germany).