Intercellular observation of connexin proteins by immunofluorescence (IF)

IL1-WT and IL1-KO cells were cultured on cover slips and were allowed to attach for 24 h before exposure to 5 μg/ml of CNT-1 and CNT-2 for 24 h. Cells were then fixed for 20 min in 4 % paraformaldehyde and permeabilized with 0.1 % Triton X-100 in PBS for 5 min. Cover slips were blocked by incubation with 5 % BSA in 0.1 % PBS-Triton X-100 for 1 h at room temperature and incubated overnight with a primary antibody against Gja1 (rabbit connexin 43 polyclonal antibody, Cell Signaling Technology), Gjb1 (mouse anti-connexin-32 monoclonal antibody, Millipore) or Gjb2 (goat anti-Gjb2 polyclonal antibody, Abcam) in 3 % BSA in PBS at 4 °C in a humidified chamber. Secondary antibodies anti-rabbit Alexa Fluor 488, anti-mouse Alexa Fluor 488 or anti-goat Alexa Fluor 488 (all from Molecular Probes) were left on the cells for 1 h at room temperature in a humidified chamber. To visualize cell nuclei cells were counterstained with Hoechst (Sigma-Aldrich). Mowiol was used for mounting of the cover slips. Fluorescence was observed using a laser scanning microscope (LSM 710, Zeiss) and pictures were taken with an AxioCam camera (Zeiss).