All lanes : Anti-Vimentin antibody [SP20] (ab16700)

Lane 1 : U20S cell lysate
Lane 2 : Hu tonsil cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : VIM knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 53 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab16700 observed at 53 kDa. Red - loading control, ab8245 observed at 37 kDa.

 ab16700 was shown to react with Vimentin in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255446 (knockout cell lysate ab263775) was used. Wild-type and Vimentin knockout samples were subjected to SDS-PAGE. ab16700 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 100 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

Anti-Vimentin antibody [SP20] (ab16700) at 1/100 dilution + HeLa cell lysate

Predicted band size: 53 kDa
Observed band size: 53 kDa

ab16700 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 ab16700 at 1/1000 dilution and ab195889 at 1/250 dilution (shown in pseudo-color red) overnight at +4°C. The cells were then incubated with ab150081 (Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488)) at 1/1000 dilution for 1 hour. Nuclear DNA was labelled in blue with DAPI.

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

Immunohistochemical analysis of Human melanona tissue labelling Vimentin with ab16700.

IHC image of ab16700 staining in Breast Cancer formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16700, (Neat supernatant) for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

ab16700 staining Vimentin in human corneal limbal epithelial cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.3% Triton X-100 for 5 minutes. Samples were incubated with primary antibody (1/200 in PBS + 10% normal goat serum) for 18 hours at 4°C. An Alexa Fluor®488-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

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ab16700 at 1/200 staining Human Limbal Epithelial Cells by ICC/IF. The cells were incubated with the antibody for 1 hour and then a FITC conjugated goat antibody was used as the secondary. The image shows vimentin staining in green and hoechst staining in blue. The upper cells in the image (vimentin negative) are epithelium cells. the vimentin positive cells are stroma cells.

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Flow cytometric analysis of rabbit anti-Vimentin (SP20) antibody ab16700 (1/100) in HeLa cells (green) compared to negative control of rabbit IgG (blue).

Overlay histogram showing HeLa cells stained with ab16700 (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 (ab16700, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.