Anti cx43 polyclonal antibody

Astrocytes plated on glass coverslips were with 2% paraformaldehyde (PFA) for 30 min fixed at room temperature. After washing three times with PBS, they were rinsed three times with (5 min each) 0.1 M PBS-glycine, and then incubated with 0.1% Triton X-100 in PBS containing 10% NGS for 30 min. Then astrocytes were incubated with an anti-GFAP monoclonal antibody (BD Biosciences, 1:400) or anti-Cx43 polyclonal antibody (SIGMA, 1:400) diluted in 0.1% Triton X-100 in PBS with 2% NGS at 4 °C overnight. After five rinses in 0.1% Triton X-100 in PBS, cells were incubated with goat anti-mouse IgG Alexa Fluor 355 (1:1000) or goat anti-rabbit IgG Alexa Fluor 488 (1:1000) at room temperature for 50 min. After washing, coverslips were mounted in DAKO fluorescent mounting medium and examined with an Olympus BX 51W1I upright microscope with a 40× water immersion. Nuclei were stained with DAPI or Hoechst 33342.

Cells grown on glass coverslips were fixed at room temperature with 2% paraformaldehyde for 30 min and then washed three times with PBS. Then, cells were incubated three times for 5 min in 0.1 M PBS-glycine, followed by 30 min incubation with 0.1% PBS-Triton X-100 containing 10% NGS. The permeabilized cells were incubated with anti-β-tubulin monoclonal antibody (Sigma, 1:400) and anti-Cx43 polyclonal antibody (SIGMA, 1:400) 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 555 (1:1,000), goat anti-rabbit IgG Alexa Fluor 488 (1:1,000), or Alexa Fluor 488-phalloidin at room temperature for 50 min. After several rinses, coverslips were mounted in DAPI Fluoromount-G medium and examined with an Olympus BX 51W1I upright microscope with a 40× water immersion objective or a confocal laser-scanning microscope with a 63× objective (Olympus, Fluoview FV1000, Tokyo, Japan).

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.