ab8978 staining Vimentinin wild-type HAP1 cells (top panel) and VIMknockout 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 ab8978 at 1μg/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 MouseIgG (Alexa Fluor® 488)) 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).

Immunohistochemistry analysis of frozen section of pig colon labelingVimentin with ab8978, showing positive staining in connective tissue cells and no reactivity in epithelial cells.

Immunohistochemistry analysis of formalin fixed, paraffin embedded section of human placenta labeling Vimentinwith ab8978, showing positive staining in connective tissue cells and no reactivity in epithelial cells.

Overlay histogram showing HeLa cells stained withab8978 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Triton 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 (ab8978, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This anti-Vimentin antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Triton used under the same conditions.

Silencing of Lamin A/C in unmethylated neuroblastoma cells induces changes in different cytoskeletal components

Immunofluorescence staining showing changes in SK-N-SH-lamin-A/C-shRNA compared with SK-N-SH-scramble-shRNA in(A)β-actin filaments,(B)F-actin filaments.(C)Vimentin filaments, and(D)α-tubulin. Lamin A/C is shown in green; β-actin, F-actin, vimentin, and α-tubulin are shown in red.DNA is stained in blue (DAPI). Scale bar, 10μM.

Vimentin is detected using ab8978 at 1/100 dilution. Neuroblastoma cells were fixed with 4% paraformaldehyde and permeabilized using 0.5% Triton X-100.

(From Figure 5C of Rauschert et al)

Immunohistochemistry analysis of formalin fixed, paraffin embedded section of human small intestine labeling Vimentin with ab8978; showing positive staining in connective tissue cells and no reactivity in epithelial cells.

Immunohistochemistry analysis of frozen section of pig colon labelingVimentin with ab8978, showing positive staining in connective tissue cells and no reactivity in epithelial cells.Nuclear staining with DAPI.

Immunohistochemistry analysis of frozen section of zebrafish embryo labeling Vimentinwith ab8978.

Immunohistochemistry analysis of formalin fixed, paraffin embedded section of human spleen labeling Vimentinwith ab8978, showing positive staining in connective tissue cells and lymphoid cells.

Immunohistochemistry analysis of frozen section of zebrafish embryo labelingVimentinwith ab8978.

Paraffin-embedded humancolontissue stained forVimentin using ab8978 at 1/100 dilution in immunohistochemical analysis.

ab8978 stainingVimentin in human fetal kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with CAS-Block for1 hour at 25°C; antigen retrieval was by heat mediation using OmniPrep (pH 9). Samples were incubated with primary antibody (1/500) for 1 hour at 25°C. An Alexa Fluor®555-conjugated Donkey polyclonal (1/200) was used as the secondary antibody.

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Immunofluorescence staining images of 9 day old zebrafish embryos. ab8978 reacts with in connective tissue cells and bloodvessels. Frozen sample treated with Acetone:Methanol 1:1, antibody diluted 1/100 and incubated for 45 minutes at room temperature.

ab8978 staining Vimentin in human colon fibroblasts by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with methanol, permeabilized in 0.1% Triton and blocked with 0.25% serum free protein blocker for 20 minutes at 28°C. Samples were incubated with primary antibody (1/100 in antibody diluent) for 2 hours at 28°C. ab6785 Goat polyclonal anti-Mouse IgG - H&L (FITC) (1/800) was used as the secondary antibody. Nuclei were counterstained with propidium iodide.

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ab8978 stainingVimentin in dogsoft tissue sarcoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 15% serum for1 hourat 20°C; antigen retrieval was by heat mediation in aTris/EDTA pH9buffer. Samples were incubated with primary antibody (1/100 in TBS) for18 hours at 20°C. A Alexa Fluor® 647-conjugatedGoat anti-mouse IgG polyclonal (1/400) was used as the secondary antibody.

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IHC-Fr image of Ed18 ratstained with ab8978. Fresh frozen sections were incubated in 10% normal donkey serum in 0.1% PBS- and 0.3% triton X100 for 1h to permeabilise the tissues and block non-specific protein-protein interactions. The sectons were then incubated with the ab8978 (1µg/ml) and ferroportin overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 donkey anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 568 (red) donkey anti-mouse at a 1/1000 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. Vimentin expressed in the gut muscles.

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Immunohistochemistry analysis of formalin fixed, paraffin embedded section of human tonsillar lymphoma labelingVimentin with ab8978.

IHC-FoFr image of vimentin staining on rat injured cortical sections using ab8978 (1:500). The brain was perfusion fixed using 4% PFA and the sections were permeabilized using 0.1% TritonX in 0.1% PBS. The sections were then blocked using 10% donkey serum for 1 hour at 24°C. ab8978 was diluted 1:500 and incubated with sections for 24 hours using 4°C. The secondary antibody used was donkey polyclonal to rabbit IgG conjugated to Alexa Fluor 488.

ab8978 staining vimentin in human pancreatic adenocarcinoma cells by immunocytochemistry/ immunofluorescence. Cells were PFA fixed and permeabilized in 0.2% Triton X prior to blocking in 3% BSA for 30 minutes at 24°C. The primary antibody was diluted 1/200 and incubated with the sample for 16 hours at 21°C. Alexa fluor® 488 mouse polyclonal to mouse Ig, diluted 1/300 was used as the secondary antibody.

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ab8978 stainingVimentininhuman lungtissue section by Immunohistochemistry (Frozen sections). Tissue samples were fixed with formaldehyde and blocking with 5%commercially available blocking agent was performed at 37⁰Cfor 15 minutes. The sample was incubated with primary antibody (1/250) at 37⁰Cfor1 hour. A HRP-conjugated Goat polyclonal to mouse IgG was used as secondary antibody at 1/1000 dilution.

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