Paramount dapi fluorescent mounting medium

MCs grown on coverslips were fixed at RT with 2% paraformaldehyde for 30 min and then washed three times with PBS. They were incubated three times for 5 min in 0.1 M PBS glycine, and then in 0.1% PBS-Triton X-100 containing 10% NGS for 30 min. Cells were incubated with anti-PDGFRB monoclonal antibody (Invitrogen, 1:100) and anti-Cx43 polyclonal antibody (#C6219 SIGMA, 1:100) diluted in 0.1% PBS-Triton X-100 with 2% NGS at 4 °C overnight. After five rinses in 0.1% PBS-Triton X-100, cells were incubated with goat anti-mouse IgG Alexa Fluor 488 (1:1000) or goat anti-rabbit IgG Alexa Fluor 555 (1:1000) at RT for 60 min. After several rinses, coverslips were mounted in Paramount-DAPI fluorescent mounting medium and examined with high-resolution fluorescence microscopy (Leica, Wetzlar, Germany) with a 63X objective.

Mesangial cells grown on coverslips were fixed at room temperature (RT) with 2% paraformaldehyde for 30 min and then washed three times with PBS. They were incubated three times for 5 min in 0.1 M PBS glycine and then in 0.1% PBS-Triton X-100 containing 10% NGS for 30 min. Cells were incubated with anti-Panx1 polyclonal antibody (1:100) diluted in 0.1% PBS-Triton X-100 with 2% NGS at 4°C overnight. After five rinses in 0.1% PBS-Triton X-100, cells were incubated with goat anti-rabbit IgG Alexa Fluor 555 (1:1,000) at RT for 60 min. After several rinses, coverslips were mounted in Paramount-DAPI fluorescent mounting medium and examined with high-resolution fluorescence microscopy (Leica, Wetzlar, Germany) with a 63X objective.