Alexa 594 conjugated goat anti rabbit mouse iggs

Cells grown or differentiated on round glass coverslips in 24-well plates were fixed and permeabilized with 100% cold methanol for 10 min. Fixed cells were incubated for 1 h in PBS containing 3% bovine serum albumin for blocking, followed by 2 h of incubation with specific primary antibodies. Cells were washed three times with TBS-T, then incubated with Cy2-conjugated goat anti-rabbit/mouse IgGs (Jackson ImmunoResearch Laboratories) or Alexa 594-conjugated goat anti-rabbit/mouse IgGs (Molecular Probes, Eugene, OR, USA) as required according to the primary antibody. Cellular DNA was counterstained with 4′,6′-diamidino-2-phenylindole (0.2 μg/mL in PBS).

Cells grown or differentiated on round glass coverslips in 24-well plates were fixed and permeabilized with 100% cold methanol for 10 min. Fixed cells were incubated for 1 h in phosphate-buffered saline with Tween 20 (PBST; PBS + 0.1% Tween 20) containing 3% bovine serum albumin for blocking, followed by incubation for 2 h at 37 °C with specific primary antibodies. Anti-calponin 1 antibody (ab46794) was obtained from Abcam (Cambridge, UK) and anti-alpha-smooth muscle actin (α-SMA; a5228) antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cells were washed three times with PBST and incubated with Cy2-conjugated goat anti-rabbit/mouse IgGs (Jackson Immunoresearch Laboratories, West Grove, PA, USA) or Alexa 594-conjugated goat anti-rabbit/mouse IgGs (Molecular Probes, Eugene, OR, USA) as required. Cellular DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 0.2 μg/mL in PBS).