Recombinant Alexa Fluor® 647 Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab194719)

ab194719 staining Vimentin in wild-type HAP1 cells (top panel) and Vimentin knockout HAP1 cells (bottom panel). 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 with ab194719 at 1/1000 dilution (shown in red) and ab195887 at 1/250 dilution (shown in green) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

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

Overlay histogram showing HAP1 wildtype (green line) and HAP1-VIM  knockout cells (red line) stained with ab194719. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab194719, 0.01µg/ml dilution) for 30 min at 22°C.

A rabbit monoclonal IgG isotype control antibody  (ab199093) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-VIM knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5,000 events were collected using a 40 mW Yellow/Green laser (640nm) and 670/14 bandpass filter.

This antibody can also be used in HAP1 cells fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

ab194719 staining Vimentin in NIH3T3 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab194719 at 1/100 dilution(shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 488, shown in green) at 2µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

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

Overlay histogram showing NIH3T3 cells stained with ab194719 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 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 (ab194719, 1/500 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor^® 647 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 solid-state 25mW red diode laser (635 nm) and 675/30 bandpass filter.

This antibody gave a positive signal in NIH3T3 fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Triton X-100 for 20 min used under the same conditions.