High resolution fluorescence microscopy

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.

Apoptosis was assessed using Click-iT TUNEL Alexa Fluor Imaging Assay as recommended by the supplier (C-10246; Invitrogen). Briefly, MCs were cultured in a 5% CO₂ incubator at 37 °C. Then, cells were fixed with 4% paraformaldehyde in PBS for 1 h at RT. Fixed cells were treated with permeabilization solution (0.1% Triton X-100) for 2 min at RT and then incubated with 50 μL of TUNEL reaction buffer for 1 h in a 37 °C humidified atmosphere in the dark. After incubation, cells were treated with 50 μL of converter-POD (anti-fluorescein antibody, Fab fragment from sheep, conjugated with horse-radish peroxidase) in a 37 °C humidified chamber for 30 min and then treated with 50 μL DAB (3,3′-diaminobenzidine) substrate for 10 min at RT. Percentage of apoptotic cells was estimated by counting TUNEL-positive red cells divided by the number of ≥15 cells for field. For each trial, data were quantified by measuring fluorescence in five representative fields using high-resolution fluorescence microscopy (Leica, Wetzlar, Germany).

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.