重组Anti-Vimentin抗体[SP20] (ab16700)

Immunohistochemical analysis of paraffin-embedded Human melanoma tissue labeling Vimentin with ab16700 at 1/200 dilution. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

This image was generated using a previous batch manufactured using the hybridoma production method.

False colour image of Western blot: Anti-Vimentin antibody [SP20] staining at 1/120 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab16700 was shown to bind specifically to Vimentin. A band was observed at 55 kDa in wild-type A549 cell lysates with no signal observed at this size in VIM knockout cell line ab288984. To generate this image, wild-type and VIM knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween$®$ 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

This image was generated using a previous batch manufactured using the hybridoma production method.

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.

This image was generated using a previous batch manufactured using the hybridoma production method.

This image was generated using a previous batch manufactured using the hybridoma production method.

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).

This image was generated using a previous batch manufactured using the hybridoma production method.

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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This image was generated using a previous batch manufactured using the hybridoma production method.

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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This image was generated using a previous batch manufactured using the hybridoma production method.

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

This image was generated using a previous batch manufactured using the hybridoma production method.

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.