Recombinant Alexa Fluor® 568 Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab202504)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-VIM  knockout cells (red line) stained with ab202504. 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 (ab202504, 0.1µg/ml dilution) for 30 min at 22°C.

A rabbit monoclonal IgG isotype control antibody  (ab209613) 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 50 mW Yellow/Green laser (561nm) and 586/15 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.

ab202504 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 overnight at +4°C with ab202504 at a 1/1000 dilution (shown in green) and ab195884, Rat monoclonal [YOL1/34] to Tubulin (Alexa Fluor® 647), at a 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

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

ab202504 staining Vimentin in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), 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 ab202504 at 1/1000 dilution (pseudocolored in green) and ab195884, Rat monoclonal to Tubulin (Alexa Fluor^® 647), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

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

This product also gave a positive signal under the same testing conditions in HeLa cells fixed with 100% methanol (5 min).

ab202504 staining Vimentin in NIH3T3 cells. The cells were fixed with 4% formaldehyde (10 min), 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 ab202504 at a 1/1000 dilution (shown in pseudocolor green) and ab195884, Rat monoclonal to alpha Tubulin (Alexa Fluor^® 647), at a 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

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

This product also gave a positive signal under the same testing conditions in NIH3T3 cells fixed with 100% methanol (5 min).