ab184675 staining alpha Smooth Muscle Actin in SV40LT-SMC cells. The cells were fixed with 100% Methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab184675 at 1/250 dilution (shown in green) and ab195884, Rat monoclonal to alpha Tubulin (Alexa Fluor^® 647), at 2µg/ml (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Overlay histogram showing SV40LT-SMC cells stained with ab184675 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab184675, 1/50 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor® 488 used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This antibody gave a positive signal in SV40LT-SMC fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Emission spectra of various Alexa Fluor® conjugates