Western blot - Vimentin antibody [V9] (ab8069)

All lanes : Anti-Vimentin antibody [V9] (ab8069) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : MOLT4 (Human acute lymphoblastic leukemia cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 54 kDa
Observed band size : 57 kDa (why is the actual band size different from the predicted?)


Exposure time : 20 minutes

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Vimentin antibody [V9] (ab8069)

IHC image of Vimentin staining in human Hodgkin's Lymphoma 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 ab8069, 1µg/ml, 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

Flow Cytometry-Vimentin antibody [V9](ab8069)

Overlay histogram showing HeLa cells stained with ab8069 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100® 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 (ab8069, 1/20 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Triton X-100® used under the same conditions.

Immunocytochemistry/ Immunofluorescence - Vimentin antibody [V9] (ab8069)

ab8069 staining Vimentin in human epithelial ovarian serous adenocarcinoma cell line SKOV-3 by Immunocytochemistry/ Immunofluorescence.

Samples were fixed with 4% PFA in PBS pH 7.4 and then permeabilised using 0.2% saponin for 30 minutes. A blocking step was performed using 1% BSA/PBS for 1 hour. Samples were then incubated with ab8069 at a 1/200 dilution in 1% BSA/PBS for 1 hour. The secondary antibody was a goat anti-mouse Alexa 488 (green) diluted 1/1000, 1% BSA/PBS for 1 hour. Samples were then incubated with phalloidin (red for actin staining) in 1% BSA/PBS for 45 minutes and counterstained with DAPI (blue for nuclei staining) in PBS for 45 minutes.

Image from Loessner D et al, Biomaterials. 2010 Nov;31(32):8494-506. Epub 2010 Aug 14. doi:10.1016/j.biomaterials.2010.07.064

Immunocytochemistry/ Immunofluorescence - Anti-Vimentin antibody [V9] (ab8069)

ICC/IF image of ab8069 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab8069, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h.Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.