Vimentin Mouse Monoclonal Antibody [D4-B11]
cat.: EM0401
Product Type: Mouse monoclonal IgG1, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, IHC-P, FC, IF-Tissue, IHC-Fr, IP
Clonality: Monoclonal
Clone number: D4-B11
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*PBS (pH7.4), 0.2% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 2ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 54 kDa
Isotype: IgG1
Immunogen: Synthetic peptide within C-terminal human Vimentin.
Positive control: HeLa cell lysate, C2C12 cell lysate, C6 cell lysate, NIH/3T3 cell lysate, Mouse embryonic stem cell lysate, Hela, human kidney tissue, human colon carcinoma tissue, human stomach carcinoma tissue, human tonsil tissue, human skin tissue, human liver tissue, human appendix tissue, human endometrium tissue.
Subcellular location: Cytoplasm
Recommended Dilutions:
  WB
  IF-Cell
  IHC-P
  FC
  IF-Tissue
  IHC-Fr
  IP
1:2,000-1:5,000
1:200
1:200
1:500-1:1,000
200-400
1:100
Use at an assay dependent concentration
Uniprot #: SwissProt: P08670 Human | P20152 Mouse | P31000 Rat
Alternative names: CTRCT30 Epididymis luminal protein 113 FLJ36605 HEL113 VIM VIME_HUMAN Vimentin
Images
EM0401_1.jpg Fig1: All lanes: Western blot analysis of Vimentin with anti-Vimentin antibody [D4-B11] (EM0401) at 1:500 dilution.
Lane 1: Wild-type Hela whole cell lysate (20 µg).
Lane 2: Vimentin knockout Hela whole cell lysate (20 µg).

EM0401 was shown to specifically react with Vimentin in wild-type Hela cells. No band was observed when Vimentin knockout sample was tested. Wild-type and Vimentin knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (EM0401, 1/500) and Loading control antibody (Rabbit anti-GAPDH , ET1601-4, 1/10,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG-HRP Secondary Antibody (HA1006) at 1:20,000 dilution was used for 1 hour at room temperature.
EM0401_2.jpg Fig2: Western blot analysis of Vimentin on different lysates with Rabbit anti-Vimentin antibody (EM0401) at 1/1,000 dilution.

Lane 1: 293T cell lysate
Lane 2: HeLa cell lysate
Lane 3: Jurkat cell lysate
Lane 4: C2C12 cell lysate
Lane 5: NIH/3T3 cell lysate
Lane 6: C6 cell lysate

Lysates/proteins at 20 µg/Lane.

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

Exposure time:15 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (EM0401) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
EM0401_3.jpg Fig3: Immunofluorescence analysis of paraffin-embedded human kidney tissue labeling Vimentin (EM0401) and NPHS2 (ET7107-34).

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS. And then probed with the primary antibodies Vimentin (EM0401, green) at 1/400 dilution and NPHS2 (ET7107-34, red) at 1/100 dilution overnight at 4 ℃, washed with PBS.

iFluor™ 488 conjugate-Goat anti-Mouse IgG (HA1125) and iFluor™ 594 conjugate-Goat anti-Rabbit IgG (HA1122) were used as the secondary antibodies at 1/1,000 dilution. DAPI was used as nuclear counterstain.
EM0401_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue using anti-Vimentin antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM0401, 1/800) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
EM0401_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded human stomach carcinoma tissue with Mouse anti-Vimentin antibody (EM0401) at 1/2,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM0401) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
EM0401_6.jpg Fig6: Immunofluorescence analysis of paraffin-embedded human tonsil tissue labeling Vimentin (EM0401) at 1/200 dilution and Cytokeratin 5 (ET1610-43) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS. And then probed with the primary antibodies Vimentin (EM0401, green) at 1/200 dilution and Cytokeratin 5 (ET1610-43, red) at 1/200 dilution at +4℃ overnight, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Mouse IgG and Alexa Fluor®594 conjugate-Goat anti-Rabbit IgG were used as the secondary antibodies at 1/1,000 dilution. DAPI was used as nuclear counterstain.
EM0401_7.jpg Fig7: Immunofluorescence analysis of paraffin-embedded human skin tissue labeling Vimentin (EM0401) at 1/200 dilution and Cytokeratin 5 (ET1610-43) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS. And then probed with the primary antibodies Vimentin (EM0401, green) at 1/200 dilution and Cytokeratin 5 (ET1610-43, red) at 1/200 dilution at +4℃ overnight, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Mouse IgG and Alexa Fluor®594 conjugate-Goat anti-Rabbit IgG were used as the secondary antibodies at 1/1,000 dilution. DAPI was used as nuclear counterstain.
EM0401_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded human liver tissue with Mouse anti-Vimentin antibody (EM0401) at 1/2,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM0401) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
EM0401_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded human appendix tissue with Mouse anti-Vimentin antibody (EM0401) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM0401) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
EM0401_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Mouse anti-Vimentin antibody (EM0401) at 1/2,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM0401) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
EM0401_11.jpg Fig11: Immunohistochemical analysis of paraffin-embedded human endometrium tissue with Mouse anti-Vimentin antibody (EM0401) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM0401) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
EM0401_12.jpg Fig12: Flow cytometric analysis of Hela cells labeling Vimentin.

Cells were fixed and permeabilized.Then stained with the primary antibody (EM0401, 1ug/ml) (red) compared with Mouse IgG1 Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Mouse IgG Secondary antibody (HA1125) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
EM0401_13.jpg Fig13: Immunofluorescence analysis of frozen mouse hippocampus tissue labeling Vimentin with Rabbit anti-Vimentin antibody (EM0401).
The tissues were blocked in 3% BSA for 30 minutes at room temperature, washed with PBS, and then probed with the primary antibody (EM0401, green) at 1/100 dilution overnight at 4℃, washed with PBS. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) was used as the secondary antibody at 1/200 dilution. Nuclei were counterstained with DAPI (blue). Image acquisition was performed with KFBIO KF-FL-400 Scanner.
EM0401_14.jpg Fig14: Immunofluorescence analysis of frozen mouse cerebral cortex tissue labeling Vimentin with Rabbit anti-Vimentin antibody (EM0401).
The tissues were blocked in 3% BSA for 30 minutes at room temperature, washed with PBS, and then probed with the primary antibody (EM0401, green) at 1/100 dilution overnight at 4℃, washed with PBS. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) was used as the secondary antibody at 1/200 dilution. Nuclei were counterstained with DAPI (blue). Image acquisition was performed with KFBIO KF-FL-400 Scanner.
EM0401_15.jpg Fig15: Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-Vimentin antibody (EM0401) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM0401) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
EM0401_16.jpg Fig16: Immunohistochemical analysis of paraffin-embedded rat colon tissue with Rabbit anti-Vimentin antibody (EM0401) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM0401) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
EM0401_17.jpg Fig17: Immunocytochemistry analysis of HeLa cells labeling Vimentin with Rabbit anti-Vimentin antibody (EM0401) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Vimentin antibody (EM0401) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
EM0401_18.jpg Fig18: Immunocytochemistry analysis of C2C12 cells labeling Vimentin with Rabbit anti-Vimentin antibody (EM0401) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Vimentin antibody (EM0401) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
EM0401_19.jpg Fig19: Immunocytochemistry analysis of C6 cells labeling Vimentin with Rabbit anti-Vimentin antibody (EM0401) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Vimentin antibody (EM0401) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
EM0401_20.jpg Fig20: Vimentin was immunoprecipitated in 0.2mg HeLa cell lysate with EM0401 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using EM0401 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HeLa cell lysate (input)
Lane 2: EM0401 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of EM0401 in HeLa cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 3 mintues
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.