Alexa fluor 633 conjugated goat anti mouse secondary antibody

Cultured parietal cells treated as described above were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. After fixation, the cells were washed three times with PBS, and permeabilized with 0.3% Triton X-100 in PBS (2% BSA) for 20 min followed by incubation in PBS (2% BSA) for another 30 min. The cells were washed 3 times with PBS. Subsequently, cells were incubated with a primary mouse monoclonal anti-H⁺/K⁺ ATPase β-subunit antibody (1/200; Abcam Ltd, Cambridge, UK) and a polyclonal rabbit anti-H. pylori antibody (1/320; Dako, Glostrup, Denmark) for 1 h at 37 °C, followed by an Alexa Fluor 633-conjugated goat anti-mouse secondary antibody (1/200; Invitrogen) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1/100; Invitrogen) for 1 h at 37 °C. All antibodies were diluted in PBS and the cells were washed 5 times after incubation with the primary and secondary antibodies. Incubation for 15 min with DAPI (0.5 μg/mL; Sigma) was performed to counterstain the nuclei and the cells were rinsed 5 times in PBS. Stained cells were mounted in ProLong ^® Gold antifade reagent medium (Invitrogen) and imaged by an Olympus BX61 fluorescence microscope (Olympus Belgium N.V.).