Anti-Vimentin antibody - Cytoskeleton Marker (ab45939)

Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab45939 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
IHC-P Use a concentration of 0.1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 54 kDa (predicted molecular weight: 54 kDa).
ICC/IF Use a concentration of 1 µg/ml.
Flow Cyt Use 0.01-0.1µg for 106 cells.
IHC-Fr 1/50.

Target

Anti-Vimentin antibody - Cytoskeleton Marker images

  • IHC image of ab45939 staining Vimentin in 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 ab45939, 0.1μg/ml working concentration, 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 secondary only 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

  • ab45939 staining Vimentin in murine kidney tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then blocked with 10% serum for 30 minutes at room temperature followed by incubation with the primary antibody at a 1/2100 dilution for 18 hours at 4°C. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/200 dilution.

    See Abreview

  • Immunohistochemical detection (formaldehyde/paraffin-embedded sections) of vimentin protein using vimentin antibody - Neural Stem Cell Marker (ab45939) on human ovarian carcinoma sections. Antigen retrieval step:Heat mediated. Blocking step: 1% BSA for 10 mins at RT°C. Ab45939 was used at 1/700 for 1 hour. Secondary Antibody: Biotin-conjugated anti rabbit IG (1/300).
  • ab45939 staining Vimentin in human squamous cell carcinoma tissue sections by IHC-Fr (Frozen sections). Tissue samples were fixed with formaldehyde and blocked with 4% milk for 20 minutes at 22°C. The sample was incubated with primary antibody (1/50 in milk) at 4°C for 18 hours. A Biotin-conjugated Goat polyclonal to rabbit IgG (1/200) was used as secondary antibody.

    See Abreview

  • ab45939 staining Vimentin in SV40LT-SMC 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 ab45939 at 1μg/ml (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • ab45939 staining Vimentin in NIH3T3 cells. 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 overnight at +4°C with ab45939 at 1μg/ml (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • ICC/IF image of ab45939 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab45939, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
  • ICC/IF image of ab45939 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 (ab45939, 1µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) 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.

  • All lanes : Anti-Vimentin antibody - Cytoskeleton Marker (ab45939) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
    Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole cell lysate
    Lane 4 : A549 (Human lung adenocarcinoma epithelial cell line) Whole cell lysate
    Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
    Lane 7 : HUVEC (Human umbilical vein endothelial cell line) Whole cell lysate
    Lane 8 : A431 (Human epithelial carcinoma cell line) Whole cell lysate
    Lane 9 : Daudi (Human B lymphoblast) Whole cell lysate
    Lane 10 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    IRDye® 800CW Goat Anti-Rabbit at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size : 54 kDa
    Observed band size : 53 kDa (why is the actual band size different from the predicted?)

    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab45939 overnight at 4°C. The membrane was then blocked for an hour using LI-COR® blocking buffer before being incubated with ab92547 overnight at 4°C. Antibody binding was detected using the IRDye® 800CW Goat Anti-Rabbit secondary at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Odyssey® CLx Imaging System.

  • All lanes : Anti-Vimentin antibody - Cytoskeleton Marker (ab45939) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 3 : HEK293 Human embryonic kidney cell line Whole Cell Lysate
    Lane 4 : Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size : 54 kDa
    Observed band size : 54 kDa
  • All lanes : Anti-Vimentin antibody - Cytoskeleton Marker (ab45939) at 1 µg/ml

    Lane 1 : Recombinant Human Vimentin protein (ab73843) at 0.1 µg
    Lane 2 : Recombinant Human Vimentin protein (ab73843) at 0.01 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 54 kDa
    Observed band size : 54 kDa


    Exposure time : 10 seconds
    Ab45939 recognizes full length recombinant Human vimentin (ab73843) which has an expected molecular weight of 54 kDa.
  • Overlay histogram showing MDA-MB-231 cells stained with ab45939 (red line). The cells were fixed with 80% 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 (ab45939, 0.01μg/1x10cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/4000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (polyclonal) (ab171870, 0.01μg/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.

  • Overlay histogram showing NIH3T3 cells stained with ab45939 (red line). The cells were fixed with 80% 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 (ab45939, 0.1μg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/4000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (polyclonal) (ab171870, 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.

  • Overlay histogram showing SV40LT-SMC cells stained with ab45939 (red line). The cells were fixed with 80% 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 (ab45939, 0.01μg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/4000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (polyclonal) (ab171870, 0.01μg/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.


  • developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 54 kDa
    Ab45939 recognizes full length recombinant Human vimentin (ab73843) which has an expected molecular weight of 54 kDa.

References for Anti-Vimentin antibody - Cytoskeleton Marker (ab45939)

This product has been referenced in:
  • Hickman GJ  et al. Controlling the dynamics of cell transition in heterogeneous cultures using surface chemistry. Adv Healthc Mater 4:593-601 (2015). Read more (PubMed: 25393206) »
  • Robertson MJ  et al. Optimizing recellularization of whole decellularized heart extracellular matrix. PLoS One 9:e90406 (2014). IHC-P ; Rat . Read more (PubMed: 24587354) »

See all 19 Publications for this product

Product Wall

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Blockaid molecular probes as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Antigen retrieval step None
Sample Sheep Tissue sections (Lung)
Specification Lung
Permeabilization No
Fixative Zinc Salts
Username

Helen Todd

Verified customer

Submitted Aug 18 2014

Application Western blot
Loading amount 40 µg
Gel Running Conditions Reduced Denaturing (12% page)
Sample Human Cell lysate - whole cell (hep3b and L3.6PL)
Specification hep3b and L3.6PL
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5%
Username

Abcam user community

Verified customer

Submitted Jan 16 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 4°C
Sample Human Cell (human ascites- pancreatic cancer cell line)
Specification human ascites- pancreatic cancer cell line
Permeabilization No
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jan 14 2014

Thank you for contacting us. Because we carry over 90,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guaran...

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Abcam has not validated the combination of species/application used in this Abreview.
Application Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample Rat Tissue sections (Brain)
Specification Brain
Fixative Paraformaldehyde
Permeabilization No
Username

Dr. Sophie Pezet

Verified customer

Submitted Mar 20 2012

Anbei wie versprochen die Publikationen aus der Gruppe von Gabbiani bzw. Bochaton-Piallat (PMIDs 21868702 bzw. 17347479). Ich hoffe, diese helfen Ihnen zumindest als Ausgangspunkt für Ihre weitere Suche nach geeigneten Markern. Vielleicht ist einer von...

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Application Western blot
Sample Human Recombinant protein (recombinant vimentin)
Loading amount 0.01 µg
Specification recombinant vimentin
Gel Running Conditions Reduced Denaturing
Blocking step Amersham ECL Blocking Agent as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Aug 18 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (Kidney)
Specification Kidney
Fixative Formaldehyde
Antigen retrieval step Heat mediated
Permeabilization No
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: RT°C
Username

Mike Forbes

Verified customer

Submitted Feb 09 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Human Tissue sections (squamous cell carcinoma)
Specification squamous cell carcinoma
Fixative Formaldehyde
Permeabilization No
Blocking step Milk as blocking agent for 20 minute(s) · Concentration: 4% · Temperature: 22°C
Username

Abcam user community

Verified customer

Submitted Mar 01 2010

Abcam has not validated the combination of species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Monkey Tissue sections (Cerebellum)
Specification Cerebellum
Fixative Formaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citric acid ph6
Permeabilization No
Blocking step BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: rt°C
Username

Mr. Carl Hobbs

Verified customer

Submitted Dec 21 2009

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"