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Recombinant full length protein corresponding to Human Vimentin.
Our Abpromise guarantee covers the use of ab3974 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use a concentration of 1 - 10 µg/ml.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
|Sandwich ELISA||Use at an assay dependent concentration.|
|ELISA||Use at an assay dependent concentration.|
|ICC||Use a concentration of 5 - 10 µg/ml.|
|WB||Use a concentration of 1 - 2 µg/ml. Use under non reducing condition. Detects a band of approximately 57 kDa. Positive control: LEP-19 cell lysate
Negative control: 3T3 mouse cell line Sample preparation: Resuspend approx. 50 mil. cells in 1 ml cold Lysis buffer (1% laurylmaltoside in 20 mM Tris/Cl, 100 mM NaCl pH 8.2, 50 mM NaF including Protease inhibitor Cocktail). Incubate 60 min on ice. Centrifuge to remove cell debris. Mix lysate with reducing Laemmli SDS-PAGE sample buffer. Boil for 3 min in water bath.
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
IHC image of Vimentin staining in human bladder formalin fixed paraffin embedded tissue section, performed on a Leica Bond 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 ab3974, 5µg/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.
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.
ICC image of ab3974 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 (ab3974, 5µg/ml) overnight at +4°C. 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.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"