All lanes : Anti-Vimentin antibody [VI-10] (ab20346) at 1 µg/ml

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : VIM (Vimentin) knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 54 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab20346 observed at 57 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab20346 was shown to specifically react with Vimentin in wild-type HAP1 cells as signal was lost in VIM (Vimentin) knockout cells. Wild-type and VIM (Vimentin) knockout samples were subjected to SDS-PAGE. Ab20346 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ab20346 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 ab20346 at 1μg/ml dilution and ab202272 at 1/250 dilution (shown in pseudo-color red) overnight at +4°C. The cells were then incubated with ab150117 (Goat Anti-Mouse 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 formalin-fixed, paraffin-embedded human skin tissue labeling Vimentin with ab20346 at 5 ug/ml.

Immunocytochemical analysis of chicken postnatal erythrocytes labeling Vimentin with ab20346 at 1 ug/ml (green). Cells were counterstained with anti-alpha-tubulin (red) and DAPI (blue).

ab20346 at a 1/500 dilution staining mouse dissociated neural precursor cells by immunocytochemistry. The antibody was incubated with the cells for 1 hour and then detected using a goat anti-mouse Alexa-Fluor 568 (red stain) ab175473>ab175473). The image also shows a blue nuclear counterstain. Confocal image is shown with 2x zoom detail in top right corner.

This image is courtesy of an Abreview submitted by Randal Moldich on 22 February 2006.

See Abreview

Human normal skin. Staining is localised to cytoplasm. Left panel: with primary antibody at 1 ug/mL. Right panel: isotype control. Sections were stained using an automated system DAKO Autostainer Plus , at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for mouse for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

ab20346 used at a 1/1000 dilution in Western Blot.5-20µg of total protein from each sample was loaded.

Overlay histogram showing NIH3T3 cells stained with ab20346 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Triton 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 (ab20346, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgM [ICIGM] (ab91545, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in NIH3T3 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Triton used under the same conditions.

Immunofluorescence staining of RBL rat basophiilioc cell line with anti-Vimentin (VI--10). Nuclei are stained with DAPI (blue).