Anti-Vimentin antibody [V9] - Cytoskeleton Marker (ab8069)

Overview

  • Product name
    Anti-Vimentin antibody [V9] - Cytoskeleton Marker
    See all Vimentin primary antibodies
  • Description
    Mouse monoclonal [V9] to Vimentin - Cytoskeleton Marker
  • Specificity
    Does not react with GFAP, neurofilamen or desmin.
  • Tested applications
    Suitable for: ICC/IF, Flow Cyt, IHC-FoFr, IHC-P, IHC-Fr, WBmore details
  • Species reactivity
    Reacts with: Rat, Horse, Chicken, Cow, Cat, Dog, Human, Pig
    Does not react with: Mouse
  • Immunogen

    Porcine Lens

  • Positive control
  • General notes

    This antibody clone is manufactured by Abcam.

    This monoclonal antibody to vimentin has been knockout validated in ICC/IF. The expected staining was observed in wild type cells and no staining was seen in vimentin knockout cells.

    Alternative versions available:

    Anti-Vimentin antibody (Alexa Fluor® 488) [V9] - Cytoskeleton Marker (ab195877)

    Anti-Vimentin antibody (Alexa Fluor® 647) [V9] - Cytoskeleton Marker (ab195878)

    Anti-Vimentin antibody (HRP) [V9] - Cytoskeleton Marker (ab196602)

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab8069 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use a concentration of 0.5 - 5 µg/ml.
Flow Cyt Use 0.1-1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

IHC-FoFr Use at an assay dependent concentration.
IHC-P Use a concentration of 0.5 - 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-Fr 1/300.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 57 kDa (predicted molecular weight: 54 kDa).

Target

  • Function
    Vimentins are class-III intermediate filaments found in various non-epithelial cells, especially mesenchymal cells. Vimentin is attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally.
    Involved with LARP6 in the stabilization of type I collagen mRNAs for CO1A1 and CO1A2.
  • Tissue specificity
    Highly expressed in fibroblasts, some expression in T- and B-lymphocytes, and little or no expression in Burkitt's lymphoma cell lines. Expressed in many hormone-independent mammary carcinoma cell lines.
  • Involvement in disease
    Cataract 30
  • Sequence similarities
    Belongs to the intermediate filament family.
  • Domain
    The central alpha-helical coiled-coil rod region mediates elementary homodimerization.
    The [IL]-x-C-x-x-[DE] motif is a proposed target motif for cysteine S-nitrosylation mediated by the iNOS-S100A8/A9 transnitrosylase complex.
  • Post-translational
    modifications
    Filament disassembly during mitosis is promoted by phosphorylation at Ser-55 as well as by nestin (By similarity). One of the most prominent phosphoproteins in various cells of mesenchymal origin. Phosphorylation is enhanced during cell division, at which time vimentin filaments are significantly reorganized. Phosphorylation by PKN1 inhibits the formation of filaments. Phosphorylated at Ser-56 by CDK5 during neutrophil secretion in the cytoplasm. Phosphorylated by STK33.
    O-glycosylated during cytokinesis at sites identical or close to phosphorylation sites, this interferes with the phosphorylation status.
    S-nitrosylation is induced by interferon-gamma and oxidatively-modified low-densitity lipoprotein (LDL(ox)) possibly implicating the iNOS-S100A8/9 transnitrosylase complex.
  • Cellular localization
    Cytoplasm.
  • Information by UniProt
  • Database links
  • Form
    Vimentin is found in connective tissue and in the cytoskeleton.
  • Alternative names
    • CTRCT30 antibody
    • Epididymis luminal protein 113 antibody
    • FLJ36605 antibody
    • HEL113 antibody
    • VIM antibody
    • VIME_HUMAN antibody
    • Vimentin antibody
    see all

Images

  • ab8069 staining Vimentin (colored green) in wild-type HAP1 cells (top panel) and VIM 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 ab8069 at 0.5μg/ml and ab202272 (Rabbit monoclonal [EP1332Y] to alpha Tubulin (Alexa Fluor® 594)) at 1/250 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with ab150117 (Goat secondary antibody to Mouse IgG (Alexa Fluor® 488)) at 2 μg/ml (colored green). Nuclear DNA was labelled in blue with DAPI.

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

  • IHC image of ab8069 staining Vimentin in normal human kidney formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond. 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, 1/500 dilution, 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. No primary antibody was used in the negative control (shown on the inset).

    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.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • ab8069 staining Vimentin in HeLa cells. The cells were fixed with 4% formaldehyde (10min), 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 overnight at +4°C with ab8069 at 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at 2µg/ml (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

    Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) Whole Cell Lysate
    Lane 2 : MOLT4 (Human lymphoblastic leukemia cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) preadsorbed (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
  • Overlay histogram showing SV40LT-SMC cells stained with ab8069 (red line). The cells were fixed with 4% formaldehyde (10 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, 0.1μg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

    Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

    This antibody gave a positive signal in SV40LT-SMC cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Triton X-100 for 20 min used under the same conditions.

  • ab8069 staining Vimentin in Human oral cavity tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

    Tissue was fixed with paraformaldehyde and blocked with ab64226 Protein Block for 5 minutes at 25°C; antigen retrieval was by heat mediation in pH 6 buffer . Samples were incubated with primary antibody (1/1000 in 10% NGS) for 16 hours at 4°C. An undiluted biotinylated goat anti-rabbit polyclonal IgG was used as the secondary antibody.

    See Abreview

  • IHC image of Vimentin staining in human Hodgkin's Lymphoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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
  • 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.
  • ab8069 staining Vimentin in pig cardiac fibroblasts by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with acetone and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/100 in PBS pH 7.4) for 30 minutes. A FITC-conjugated goat anti-mouse IgG polyclonal (1/20) was used as the secondary antibody.

    See Abreview

  • ab8069 staining Vimentin in rat glial cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde and blocked with 1% BSA for 10 minutes at 25°C. Samples were incubated with primary antibody (1/200) for 10 hours at 4°C. An Alexa Fluor® 555-conjugated anti-mouse IgG polyclonal (1/300) was used as the secondary antibody.

    See Abreview

  • 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 +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.

  • Overlay histogram showing MDA-MB-231 cells stained with ab8069 (red line). The cells were fixed with 4% formaldehyde (10 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, 1μg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

    Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

    This antibody gave a positive signal in MDA-MB-231 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Triton X-100 for 20 min used under the same conditions.

References

This product has been referenced in:
  • Lee A  et al. Hair growth promoting effect of dermal papilla like tissues from canine adipose-derived mesenchymal stem cells through vascular endothelial growth factor. J Vet Med Sci 78:1811-1818 (2017). IHC ; Dog . Read more (PubMed: 27647656) »
  • Lou Y  et al. Estradiol Suppresses TLR4-triggered Apoptosis of Decidual Stromal Cells and Drives an Anti-inflammatory TH2 Shift by Activating SGK1. Int J Biol Sci 13:434-448 (2017). Read more (PubMed: 28529452) »

See all 39 Publications for this product

Customer reviews and Q&As

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Kidney)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer (pH 6)
Permeabilization
No
Specification
Kidney
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
Fixative
Paraformaldehyde
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Verified customer

Submitted Aug 02 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Dog Tissue sections (canine soft tissue sarcoma)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris/EDTA pH 9
Permeabilization
No
Specification
canine soft tissue sarcoma
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 15% · Temperature: 20°C
Fixative
Formaldehyde
Username

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Verified customer

Submitted Dec 18 2015

Application
Western blot
Loading amount
10 µg
Gel Running Conditions
Reduced Denaturing (10%)
Sample
Pig Tissue lysate - whole (Heart)
Specification
Heart
Treatment
5 pig are treated with pacemaker for 14 days (pm)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Abcam user community

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Submitted Jan 21 2015

Application
IHC - Wholemount
Sample
Human Tissue (Brain)
Specification
Brain
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Abcam user community

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Submitted Jan 06 2015

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (12% Gel)
Sample
Rat Cell lysate - whole cell (mixed glial cells)
Specification
mixed glial cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Submitted Dec 05 2014

Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (Glial Cells)
Specification
Glial Cells
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
Formaldehyde
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Submitted Jul 14 2014

Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: CItrate Buffer
Sample
Rat Tissue sections (brain)
Specification
brain
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Formaldehyde
Username

Dr. Yasir Ahmed Syed

Verified customer

Submitted Jul 09 2014

Application
Western blot
Loading amount
10 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Tissue lysate - whole (Heart)
Specification
Heart
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Jan 22 2014

Application
Immunocytochemistry/ Immunofluorescence
Sample
Pig Cell (cardiac fibroblasts)
Specification
cardiac fibroblasts
Permeabilization
Yes - Triton X-100 0.1%
Fixative
Acetone
Username

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Verified customer

Submitted Jan 09 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (oral squamous cell carcinoma)
Specification
oral squamous cell carcinoma
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Sep 10 2013

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