Anti-Vimentin antibody [EPR3776] ab92547 is a rabbit monoclonal antibody that is used in Vimentin western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity confirmed with VIM knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Antibody clone EPR3776 is cited in over 2260 publications
New 20 ul size available
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
mIHC | IHC-P | WB | ICC/IF | Flow Cyt (Intra) | |
---|---|---|---|---|---|
Human | Tested | Tested | Tested | Tested | Tested |
Mouse | Expected | Tested | Tested | Expected | Expected |
Rat | Expected | Tested | Tested | Expected | Expected |
African green monkey | Expected | Expected | Tested | Expected | Expected |
Cat | Predicted | Predicted | Predicted | Predicted | Predicted |
Pig | Predicted | Predicted | Predicted | Predicted | Predicted |
Rhesus monkey | Predicted | Predicted | Predicted | Predicted | Predicted |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat, African green monkey | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Rhesus monkey, Pig, Cat | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/200 - 1/500 | Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Species Rat | Dilution info 1/200 - 1/500 | Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Species Human | Dilution info 1/200 - 1/500 | Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Species | Dilution info | Notes |
---|---|---|
Species African green monkey | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Rhesus monkey, Pig, Cat | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/1000 - 1/5000 | Notes - |
Species Rat | Dilution info 1/1000 - 1/5000 | Notes - |
Species African green monkey | Dilution info 1/1000 - 1/5000 | Notes - |
Species Human | Dilution info 1/1000 - 1/5000 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Rhesus monkey, Pig, Cat | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 2 µg/mL | Notes This product gave a positive signal in HeLa (VIM knockout HeLa cells were used as a negative control) fixed with 4% formaldehyde (10 min) and 100% methanol (5 min). |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat, African green monkey | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Rhesus monkey, Pig, Cat | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/50 - 1/500 | Notes Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat, African green monkey | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Rhesus monkey, Pig, Cat | Dilution info - | Notes - |
Select an associated product type
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. Plays a role in cell directional movement, orientation, cell sheet organization and Golgi complex polarization at the cell migration front (By similarity). Protects SCRIB from proteasomal degradation and facilitates its localization to intermediate filaments in a cell contact-mediated manner (By similarity).Involved with LARP6 in the stabilization of type I collagen mRNAs for CO1A1 and CO1A2.
Vimentin, VIM
Anti-Vimentin antibody [EPR3776] ab92547 is a rabbit monoclonal antibody that is used in Vimentin western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity confirmed with VIM knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Antibody clone EPR3776 is cited in over 2260 publications
New 20 ul size available
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
EPR3776
Affinity purification Protein A
1.1 x 10-10 M
Blue Ice
1-2 weeks
+4°C
-20°C
Upon delivery aliquot
Avoid freeze / thaw cycle
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
This product is a recombinant monoclonal antibody, which offers several advantages including:
For more information, read more on recombinant antibodies.
This supplementary information is collated from multiple sources and compiled automatically.
Vimentin also known by its scientific name VIM is a type of intermediate filament protein that forms part of the cytoskeleton. It has a molecular mass of approximately 57 kDa. Vimentin is expressed in various cell types including mesenchymal cells fibroblasts and endothelial cells. This protein maintains cell integrity and provides resistance against stress by organizing cellular architecture and anchoring organelles. Researchers often use antibodies such as anti-vimentin or anti-mouse vimentin to study its expression and functions in cell biology.
The role of vimentin involves providing structural support and participating in processes like cell division and wound healing. Vimentin forms complexes with other cytoskeletal proteins significantly impacting cell shape and mobility. Its involvement in cellular stability becomes paramount during events requiring cellular reorganization such as embryogenesis and inflammation. These functions are frequently observed using labeled antibodies like Alexa Fluor 568 and Alexa Fluor 647 for fluorescent imaging.
Vimentin plays a role in the epithelial-to-mesenchymal transition (EMT) and interacts within the TGF-beta signaling pathway. It works alongside proteins like N-cadherin and fibronectin in the EMT pathway contributing to increased cell motility and survival. This involvement positions vimentin as a control point reflecting changes in cell phenotypes under physiological and pathological conditions.
Vimentin has relevance in cancer metastasis and cardiovascular diseases. Its aberrant expression correlates with tumor progression and metastatic potential by affecting cell movement and invasion. Vimentin also associates with proteins like collagen and integrins in the context of cardiovascular pathologies influencing vascular stiffness and remodelling. By targeting vimentin researchers aim to understand intervention strategies that may mitigate these diseases.
We have tested this species and application combination and it works. It is covered by our product promise.
We have not tested this specific species and application combination in-house, but expect it will work. It is covered by our product promise.
This species and application combination has not been tested, but we predict it will work based on strong homology. However, this combination is not covered by our product promise.
We do not recommend this combination. It is not covered by our product promise.
We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.
In the unlikely event of one of our products not working as expected, you are covered by our product promise.
Full details and terms and conditions can be found here:
Terms & Conditions.
Immunostaining of formalin fixed paraffin embedded human microtissues after exposure to MTX, TAA and TGF-β1.
Formalin fixed paraffin embedded slides of HepaRG/THP-1 macrophages/hTERT-HSC microtissues were stained with Hematoxylin & Eosin (H&E) and vimentin after 14 days of treatment with MTX, TAA and TGF-β1. Microtissues were fixed in 4% PFA and embedded in 2% agarose prior to paraffinization. Microtissues showed increase in the vimentin positive cells after MTX, TAA and TGF-β1 exposure. Vimentin stainings show proliferation of stellate cells and THP-1 macrophages in the microtissues, suggesting the onset of inflammation process.
For full image see PMID 28665955.
Vimentin immunohistochemistry staining of rhesus monkey retina tissue using rabbit anti-Vimentin antibody
Immunohistochemical staining of paraffin embedded paraformaldehyde fixed rhesus monkey retina tissue with ab92547 (green) at a working dilution of 1/200. The sample was incubaded with the primary antibody fro 20 hours at 4°C in 2.5% serum. The secondary antibody used is a Goat anti-rabbit AlexaFluor 488 at 1/400. Heat mediated antigen retrieval was perfomed using citrate pH 6. Tissue was blocked with 5% serum for 1 hour 30 minutes at 25°C
Unpurified ab92547 staining vimentin in human Schlemms Canal Endothelium cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with Triton X-100 0.2% and blocked with 10% serum for 30 minutes at 20°C. Samples were incubated with primary antibody (1/200 in DPBS) for 3 hours at 20°C. An undiluted Alexa Fluor®488-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.
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 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: Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/1000 dilution
Lane 1: HeLa (Human epithelial cell line from cervix adenocarcinoma) Whole Cell Lysate at 20 µg
Lane 2: HEK293 (Human epithelial cell line from embryonic kidney) Whole Cell Lysate at 20 µg
Lane 3: Jurkat (Human T cell lymphoblast-like cell line) Whole cell lysate at 20 µg
Lane 4: A549 (Human lung carcinoma cell line) Whole cell lysate at 20 µg
Lane 5: NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 20 µg
Lane 6: PC12 (Rat adrenal gland pheochromocytoma cell line) Whole Cell Lysate at 20 µg
Lane 7: HUVEC (Human umbilical vein endothelial cell line) Whole cell lysate at 20 µg
Lane 8: A431 (Human epidermoid carcinoma cell line) Whole cell lysate at 20 µg
Lane 9: Daudi (Human Burkitt's lymphoma cell line) Whole cell lysate at 20 µg
Lane 10: Caco 2 (Human colorectal adenocarcinoma cell line) Whole Cell Lysate at 20 µg
All lanes: IRDye® 800CW Goat Anti-Rabbit at 1/10000 dilution
Performed under reducing conditions.
Predicted band size: 53 kDa
Observed band size: 53 kDa
Different batches of ab92547 were tested on HEK-293 (Human embryonic kidney epithelial cell) lysate at 0.02 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 54 kDa.
All lanes: Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547)
Predicted band size: 53 kDa
Overlay histogram showing HAP1 wildtype (green line) and HAP1-VIM knockout cells (red line) stained with ab92547. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab92547, 0.5μg/ml) for 30 min at 22°C. The secondary antibody used wasGoat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) secondary antibodyat 1/2000 dilution for 30 min at 22°C. A Rabbit IgG isotype control antibody (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-VIM knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.
Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
All lanes: Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/5000 dilution
Lane 1: HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg
Lane 2: HEK293 (Human epithelial cell line from embryonic kidney) cell lysate at 20 µg
All lanes: HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution
Predicted band size: 53 kDa
Observed band size: 54 kDa
Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
All lanes: Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/5000 dilution
Lane 1: Mouse brain lysate
Lane 2: Rat brain lysate
All lanes: HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution
Predicted band size: 53 kDa
Observed band size: 54 kDa
Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
All lanes: Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/20000 dilution
All lanes: COS-1 (African green monkey kidney fibroblast-like cell line) cell lysate at 20 µg
All lanes: HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution
Predicted band size: 53 kDa
Observed band size: 54 kDa
ab92547 staining Vimentin in rat skin tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with 10% buffered normal formalin and blocked with 5% serum for 60 minutes at 21°C; antigen retrieval was by heat mediation in a 10mM Sodium citrate buffer. Samples were incubated with primary antibody (1/200 in blocking buffer) for 12 hours at 4°C. A Cy3®-conjugated donkey anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.
Anti-vimentin (ab92547) staining in E17 rat cheek sections using immunohistochemistry (formaldehyde-fixed, paraffin-embedded sections). Heat-mediated antigen retrieval was carried out using citric acid. Samples were incubated with primary antibody (1/2000) for two hours at room temperature. A biotin-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.
Image courtesy of Mr Carl Hobbs, Kings College London.
Vimentin flow cytometry staining of HeLa cells using rabbit anti-Vimentin antibody
Overlay histogram showing HeLa cells fixed in 2% PFA and stained with purified ab92547 at a dilution of 1 in 50 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).
Vimentin immunohistochemistry staining of mouse hippocampus using rabbit anti-Vimentin antibody
Anti-vimentin (ab92547) staining in adult mouse brain (the dentate gyrus region of the hippocampus) using immunohistochemistry (formaldehyde-fixed paraffin-embedded sections). Heat-mediated antigen retrieval was carried out using citric acid. Samples were incubated with primary antibody (1/2000 dilution) for two hours at room temperature. A biotin-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.
Image courtesy of Mr Carl Hobbs Kings College London.
Vimentin immunofluorescence staining of HeLa cells using rabbit anti-Vimentin antibody
Immunofluorescence staining of HeLa (human epithelial cell line from cervix adenocarcinoma) cells with purified ab92547 at a working dilution of 1/250 counter-stained with DAPI. The secondary antibody was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody used at a dilution of 1/1000. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 a mouse anti-tubulin antibody (1/1000) was used to stain tubulin along with Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) 1/1000 shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1 purified ab92547 was used at a dilution of 1/500 followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at a dilution of 1/500. For negative control 2 Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at a dilution of 1/400.
Vimentin immunohistochemistry staining of mouse kidney using rabbit anti-Vimentin antibody
Immunohistochemical staining of paraffin embedded mouse kidney with purified ab92547 at a working dilution of 1/250. The secondary antibody used is Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) secondary antibody at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
Immunohistochemical staining of paraffin embedded human cervical carcinoma with purified ab92547 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
Vimentin immunofluorescence staining of HeLa cells using rabbit anti-Vimentin antibody
Unpurified ab92547 staining Vimentin in HeLa cells. The cells were fixed with 100% methanol (5 min) permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab92547 at a working concentration of 5μg/ml and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 594 shown in red) at 1/250 dilution overnight at +4°C followed by a further incubation at room temperature for 1h with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) secondary antibody at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Vimentin immunofluorescence staining of HeLa cells using rabbit anti-Vimentin antibody
ab92547 staining Vimentin in HeLa (human epithelial cell line from cervix adenocarcinoma) cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab92547 at 5μg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 at 1μg/ml overnight at +4°C followed by a further incubation at room temperature for 1h with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) secondary antibody at 2 μg/ml (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.
Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human cervical carcinoma tissue sections labeling Vimentin with ab92547.
Vimentin immunohistochemistry staining of human kidney using rabbit anti-Vimentin antibody
Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human kidney tissue sections labeling Vimentin with ab92547.
Vimentin immunohistochemistry staining of human colon using rabbit anti-Vimentin antibody
Fluorescent immunohistochemical analysis of paraffin-embedded human normal colon tissue using unpurified ab92547. Green- Vimentin red-PI
Vimentin immunohistochemistry staining of human kidney using rabbit anti-Vimentin antibody
Fluorescent immunohistochemical analysis of paraffin-embedded human normal kidney tissue using unpurified ab92547. Green- Vimentin red-PI.
We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody.
Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.
ab92547 staining Vimentin 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 ab92547 at 0.5μg/ml and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) secondary antibody at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
IHC image of unpurified ab92547 staining Vimentin in human breast adenocarcinoma 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 ab92547, 1/200 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
Tissue Microarrays stained for Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker using ab92547 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negaive (cross mark) staining per sample type tested. The section was incubated with ab92547 at 4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer) secondary antibody (Goat Anti-Rabbit IgG H&L (HRP polymer) ab214880).
Heat mediated antigen retrieval was performed using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0)
Vimentin immunohistochemistry staining of human kidney using rabbit anti-Vimentin antibody
Immunohistochemical analysis of formalin fixed paraffin embedded human kidney labelling Vimentin with ab92547 at a concentration of 0.5 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab92547 anti-Vimentin antibody [EPR3776] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.
Vimentin immunofluorescence staining of human limbal stem cells using rabbit anti-Vimentin antibody
Immunocytochemistry analysis of paraformaldehyde-fixed 0.5 TritonX-100 +1% BSA permeabilized human limbal stem cells staining with ab92547 at 1/100 dilution. Secondary antibody was Alexa Fluor™ 456 Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed. The cells were incubated with the primary antibody with 1%BSA + 0.1%Triton PBS for 16 hours at 4°C. Blocking was done using 1% BSA for 30 minutes at 25°C.
Vimentin immunofluorescence staining of VIM KO cells using rabbit anti-Vimentin antibody
ab92547 staining VIM in wild-type HeLa cells with negative expression in VIM knockout HeLa cells. The cells were fixed with 4% formaldehyde (10 min) permeabilised 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 ab92547 at 2 μg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488) pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed ab150119 Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647) pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.This product also work with 100% methanol (5 min) fixation under the same testing conditions.
All lanes: Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/1000 dilution
Lane 1: KO HCT116 whole cell lysate at 30 µg
Lane 2: WT HCT116 whole cell lysate at 30 µg
All lanes: IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody at 1/20000 dilution
Performed under reducing conditions.
Exposure time: 5min
4-15% gradient, denaturing conditions
All lanes: Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/1000 dilution
All lanes: Rat Hippocampal culture whole cell lysate, at 10 µg
All lanes: Alexa Fluor 680 Donkey anti-Rabbit IgG (H+L) at 1/5000 dilution
Performed under reducing conditions.
Exposure time: 7min
This data was developed using Anti-Vimentin antibody [EPR3776] - BSA and Azide free ab193555, the same antibody clone in a different buffer formulation.
Immunofluorescence analysis of human rectum adenocarcinoma labelled with Anti-Vimentin antibody [EPR3776] - BSA and Azide free ab193555. Image was acquired using Cell DIVE Multiplexed Imaging Solution (Leica Microsystems).
Formalin fixed paraffin embedded (FFPE) sections were incubated with Anti-Vimentin antibody [EPR3776] - BSA and Azide free ab193555 at 1/100 dilution, for 1h at room temperature. Nuclear DNA was labelled with DAPI (Red). Deparaffinization, antigen retrieval, biomarker labeling and imaging was performed following Cell DIVE recommended standard protocol. Cell DIVE automatically processed the image to perform autofluorescence removal, distortion correction, blank glass subtraction, flat-field correction, and stitching.
False colour image of Western blot: Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab92547 was shown to bind specifically to Vimentin. A band was observed at 55 kDa in wild-type A549 cell lysates with no signal observed at this size in VIM knockout cell line Human VIM knockout A549 cell line ab288984. To generate this image, wild-type and VIM knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: VIM knockout A549 cell lysate at 20 µg
Lane 3: Daudi cell lysate at 20 µg
Performed under reducing conditions.
Observed band size: 55 kDa
This data was developed using Anti-Vimentin antibody [EPR3776] - BSA and Azide free ab193555, the same antibody clone in a different buffer formulation.
Multiplex immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue.
Panel A: Merged staining of anti-Vimentin (Anti-Vimentin antibody [EPR3776] - BSA and Azide free ab193555; red; Opal™690), anti-CYP11A1 (Anti-CYP11A1 antibody [EPR24868-86] ab272494; cyan; Opal™520) and anti-DDX4 / MVH (Anti-DDX4 / MVH antibody [EPR24148-58] ab270534; green; Opal™570) on human testis.
Panel B: Anti-CYP11A1 stained on Leydig cells.
Panel C: Anti-DDX4 / MVH stained on all spermatogenic cell types.
Panel D: Anti-Vimentin stained on Sertoli cells and fibroblasts.
Key protocol steps: The section was incubated in three rounds of staining: in the order of Anti-Vimentin antibody [EPR3776] - BSA and Azide free ab193555 (1:2000 dilution) and Anti-CYP11A1 antibody [EPR24868-86] ab272494 (1:10000 dilution) for 30 mins, then Anti-DDX4 / MVH antibody [EPR24148-58] ab270534 (1:2000 dlilution) for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
DAPI was used as a nuclear counter stain. Opal Polymer HRP Ms + Rb was used as a secondary.
Antigen retrieval: Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Flow cytometry overlay histogram showing wild-type HAP1 (green line) and HAP1-Vimentin knockout cells stained with ab92547 (magenta line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab92547) (1x 106in 100µl at 0.04 µg/ml (1/55750)) for 30min at 22°C.
The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C
Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type HAP1 - black line, HAP1-Vimentin knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
This antibody gave a positive signal in HAP1 WT Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.
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