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) (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.

All lanes : Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/1000 dilution (unpurified)

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) Whole Cell Lysate
Lane 2 : HEK293 (Human epithelial cell line from embryonic kidney) Whole Cell Lysate
Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole cell lysate
Lane 4 : A549 (Human lung carcinoma cell line) Whole cell lysate
Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 6 : PC12 (Rat adrenal gland pheochromocytoma cell line) Whole Cell Lysate
Lane 7 : HUVEC (Human umbilical vein endothelial cell line) Whole cell lysate
Lane 8 : A431 (Human epidermoid carcinoma cell line) Whole cell lysate
Lane 9 : Daudi (Human Burkitt's lymphoma cell line) Whole cell lysate
Lane 10 : Caco 2 (Human colorectal adenocarcinoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : 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 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.

Jhy^lacZ/lacZ mice exhibit delayed radial glial to ependymal cell differentiation.

Immunohistochemical analysis of P10 lateral ventricle coronal sections from Jhy^+/+ (A, E) and Jhy^lacZ/lacZ (I, M) mice for expression of Vimentin (pink, ab92547), Glast (green) and Acα-Tub (orange) in dorsal (A-D, I-L) and ventral (E-H, M-P) brain regions. Lower right panels (D, L, H, P) represent a higher magnification view of the merged image. In Jhy^+/+, medial wall dorsal and ventral cells express the differentiated ependymal markers Vimentin (A, B, E, F) and Acα-Tub (A, D, E, H), but are negative for the radial glial marker Glast (A, C, E, G). In Jhy^lacZ/lacZ brains, some dorsal cells remain positive for the undifferentiated marker Glast (I, K), while also expressing the differentiated markers Vimentin and Acα-Tub (I, J, L). Jhy^lacZ/lacZ ventral cells express only Vimentin and Acα-Tub (M-P). The dotted line indicates the medial wall ependymal cells in (C, G, K, O). (Q-R) Graphical representation of the percentage of Glast(-)Vimentin(+)Acα-Tub(+) (black bar) and Glast(+)Vimentin(+)Acα-Tub(+) (grey bar) cells in dorsal (Q) and ventral (R) ependymal cells. MW, medial wall; LW, lateral wall; LV, lateral ventricle; * denotes p≤0.05. Scale bars: 50μm (A-P).

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 (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.

 

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

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

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 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 (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).

 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.

All lanes : Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/5000 dilution (purified)

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 2 : HEK293 (Human epithelial cell line from embryonic kidney) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

All lanes : Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/5000 dilution (purified)

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) at 1/20000 dilution (purified) + COS-1 (African green monkey kidney fibroblast-like cell line) cell lysate at 20 µg

Secondary
HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

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

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

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) (ab150077) secondary antibody, used at a dilution of 1/1000. 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 (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 (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at a dilution of 1/400.

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 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 (ab150081) secondary antibody at 2 μg/ml (shown in green) and 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.

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 ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 594, shown in red) at 1/250 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 (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).

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).

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.

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) 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.

Anti-vimentin (ab92547) staining in human ovarian cancer tissue 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.

Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human cervical carcinoma tissue sections labeling Vimentin with ab92547.

Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human kidney tissue sections labeling Vimentin with ab92547.

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.

Fluorescent immunohistochemical analysis of paraffin-embedded human normal kidney tissue using unpurified ab92547. Green- Vimentin red-PI.

Fluorescent immunohistochemical analysis of paraffin-embedded human normal colon tissue using unpurified ab92547. Green- Vimentin red-PI

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.

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Immunofluorescence staining of NIH/3T3 (mouse embryo fibroblast cell line) whole cells with ab92547 at 1/1000 dilution in PBS, 1% BSA 0,1% Triton-X-100. The secondary antibody was Alexa Fluor­ 555 Donkey Anti-Rabbit IgG, used at a dilution of 1/1500. The cells were fixed in formaldehyde and permeabilized using 0.2% Triton X 100. Then blocked with 3% BSA for 30 minutes at room temperature.

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Equilibrium dissociation constant (K_D)
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