Alexa Fluor® 488 Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Alexa Fluor® 488 Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (AB185030)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-VIM knockout cells (red line) stained with ab185030. 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 (ab185030, 0.1μg/ml dilution) for 30 min at 22°C.

A rabbit monoclonal IgG isotype control antibody (ab199091) 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 (488nm) and 630/30 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 488 Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (AB185030)

ab185030 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 ab185030 at a 1/500 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), 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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 488 Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (AB185030)

ab185030 staining Vimentin in HeLa cells. The cells were fixed with 100% methanol (5min) 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 ab185030 (Alexa Fluor^® 488) at a working dilution of 1 in 100 overnight at +4°C (shown in green). Alexa Fluor^® 350 WGA was used at a 1/200 dilution and incubated for 1h with the cells, to label plasma membranes (shown in blue). Nuclear DNA was labelled in red with 1.25 μM DRAQ5™ (ab108410).