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Vimentin clone d21h3

Manufactured by Cell Signaling Technology
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Vimentin (clone D21H3) is a monoclonal antibody product developed by Cell Signaling Technology. Vimentin is a type III intermediate filament protein that is expressed in mesenchymal cells.

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Lab products found in correlation

Snail clone c15d3, by Cell Signaling Technology (2 mentions) Alexa fluor fluorescent dye conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions) Mouse cxcl12 sdf 1 alpha quantikine elisa kit, by R&D Systems (2 mentions) Anti pan cytokeratin clone c11, by Cell Signaling Technology (2 mentions) Anti α smooth muscle actin α sma clone 1a4, by Agilent Technologies (2 mentions) Anti cd105 pe clone sn6, by Thermo Fisher Scientific (2 mentions) Anti mitochondria clone 113 1, by Merck Group (2 mentions) Ccl5 duoset elisa kit, by R&D Systems (2 mentions) Anti ki67, by Abcam (2 mentions) Il 6 elisa kit, by BD (2 mentions) Goat serum, by Agilent Technologies (1 mentions) Cd68 clone y1 82a, by BioLegend (1 mentions) Cd45 clone hi30, by BioLegend (1 mentions) Cd163 clone ghi 61, by BioLegend (1 mentions) Vectashield containing dapi, by Vector Laboratories (1 mentions) Biotin blocking system, by Agilent Technologies (1 mentions) Image pro plus software, by Media Cybernetics (1 mentions) Trans blot turbo blotting system, by Bio-Rad (1 mentions) Mini protean tgx 4 15 gel, by Bio-Rad (1 mentions) Gapdh clone d16h11, by Cell Signaling Technology (1 mentions)

6 protocols using vimentin clone d21h3

1

Immunophenotyping Synovial Tissue Sections

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Frozen synovial tissue sections were fixed in acetone and blocked with 10% goat serum (Dako), followed by incubation with the Biotin blocking system (Dako). Stainings with mAb directed against TNF (clone 52B83; Hycult Biotech), CD45 (clone HI30; BioLegend), CD55 (clone JS11; BioLegend), CD68 (clone Y1/82A; BioLegend), CD90 (clone 5E10; BioLegend), CD163 (clone GHI/61; BioLegend), and vimentin (clone D21H3; Cell Signaling Technology) were performed overnight at 4°C, followed by incubation with Alexa Fluor 488/Alexa Fluor 555–conjugated goat anti-mouse and goat anti-rabbit secondary antibodies. Slides were mounted with Vectashield containing DAPI (Vector Laboratories) and analyzed on a fluorescence imaging microscope (Leica DMRA) coupled to a charge-coupled device camera, with results analyzed using Image-Pro Plus software (Media Cybernetics, Dutch Vision Components).
Kaaij M.H., van Tok M.N., Blijdorp I.C., Ambarus C.A., Stock M., Pots D., Knaup V.L., Armaka M., Christodoulou-Vafeiadou E., van Melsen T.K., Masdar H., Eskes H.J., Yeremenko N.G., Kollias G., Schett G., Tas S.W., van Duivenvoorde L.M, & Baeten D.L. (2020). Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis. The Journal of Experimental Medicine, 217(10), e20200288.
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2

Western Blot Analysis of EMT Markers

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Protein was extracted from the cells using Pierce RIPA Buffer (Thermo Fisher Scientific, Rockford, IL, USA) with the cOmplete, EDTA‐free Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany). A total of 20 μg whole cell extracts was loaded on mini protean TGX 4–15% gels (Bio‐Rad, Hercules, CA, USA) and transferred using the Trans‐Blot Turbo Blotting System (Bio‐Rad). The membranes were probed with the following primary antibodies: mAbs for E‐cadherin (clone 24E10; Cell Signaling Technology, Beverly, MA, USA), vimentin (clone D21H3; Cell Signaling Technology), Snail (clone C15D3; Cell Signaling Technology), SERPINI1 (clone 1D10; Sigma‐Aldrich), CHST11 (clone 1H3; Sigma‐Aldrich), or GAPDH (clone D16H11; Cell Signaling Technology) as a control at 4°C overnight. The secondary antibodies were peroxidase‐coupled goat anti‐rabbit or anti‐mouse antibodies and detected with Clarity Western ECL Substrate (Bio‐Rad), and the protein bands were visualized using the ImageQuant LAS 4000 mini system (GE Healthcare Life Sciences).
Matsuda Y., Miura K., Yamane J., Shima H., Fujibuchi W., Ishida K., Fujishima F., Ohnuma S., Sasaki H., Nagao M., Tanaka N., Satoh K., Naitoh T, & Unno M. (2016). SERPINI1 regulates epithelial–mesenchymal transition in an orthotopic implantation model of colorectal cancer. Cancer Science, 107(5), 619-628.
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3

Multiparametric Characterization of Cellular Phenotypes

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Antibodies used for staining included: anti-pan-cytokeratin (clone C11, 1:500) and vimentin (clone D21H3, 1:200) (Cell Signaling), anti- α-smooth muscle actin (α-SMA) (clone 1A4, 1:200) (Dako), anti-Ki-67 (rabbit polyclonal, ab15580, 1:300) (Abcam), anti-CD105-PE (clone SN6, 1:100) (eBioscience), anti-mitochondria (clone 113-1, 1:100) and anti-fibroblast specific protein-1 (FSP-1; rabbit polyclonal, 07-2274, 1:200) (Millipore). IL-6 ELISA kit was purchased from BD Biosciences. CCL5 DuoSet ELISA kit and mouse CXCL12/SDF-1 alpha Quantikine ELISA Kit were from R&D Systems. Alexa Fluor® fluorescent dye-conjugated secondary antibodies were from Life Technologies for immunofluorescent staining.
Shen K., Luk S., Hicks D.F., Elman J.S., Bohr S., Iwamoto Y., Murray R., Pena K., Wang F., Seker E., Weissleder R., Yarmush M.L., Toner M., Sgroi D, & Parekkadan B. (2014). Resolving Cancer-Stroma Interfacial Signaling and Interventions with Micropatterned Tumor-Stromal Assays. Nature communications, 5, 5662.
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4

Immunohistochemical Profiling of SCLC Subtypes

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TMAs and slides were stained for subtype‐defined markers of SCLC including ASCL1 (clone 24B72D11.1, dilution 1:100; BD Biosciences), NEUROD1 (clone EPR17084, dilution 1:50; Abcam), POU2F3 (clone 6D1, dilution 1:100; Santa Cruz), YAP1 (clone 63.7, dilution 1:2000; Santa Cruz) and Vimentin (clone D21H3, dilution 1:100; Cell Signaling Technologies) using Anti‐mouse/rabbit IHC Detection Kit (PK10006; Proteintech) according the manufacturer's protocol. Two independent pathologists reviewed the stained slides in a blinded fashion. The expression of each marker from tumor cells was assessed by histoscore (H‐score, range 0–300), which was calculated by multiplying the proportion of positive tumor cells (0%–100%) by the intensity of positive staining (no staining = 0, weak staining = 1, moderate staining = 2, and strong staining = 3).37, 38 A dominant marker is defined as the marker with the highest H‐score. In SCLC with combined SCLC and NSCLC components, IHC scores reflect expression exclusively in the SCLC component.
Deng C., Wang Y., Fu F., Li D., Zheng Q., Jin Y., Li Y., Chen H, & Zhang Y. (2023). Tumor‐derived Vimentin as a novel biomarker for distinct subtypes predicting adjuvant chemotherapy resistance and T‐cell‐inflamed phenotype in small cell lung cancer. MedComm, 4(5), e370.
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5

Multiparametric Characterization of Cellular Phenotypes

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Antibodies used for staining included: anti-pan-cytokeratin (clone C11, 1:500) and vimentin (clone D21H3, 1:200) (Cell Signaling), anti- α-smooth muscle actin (α-SMA) (clone 1A4, 1:200) (Dako), anti-Ki-67 (rabbit polyclonal, ab15580, 1:300) (Abcam), anti-CD105-PE (clone SN6, 1:100) (eBioscience), anti-mitochondria (clone 113-1, 1:100) and anti-fibroblast specific protein-1 (FSP-1; rabbit polyclonal, 07-2274, 1:200) (Millipore). IL-6 ELISA kit was purchased from BD Biosciences. CCL5 DuoSet ELISA kit and mouse CXCL12/SDF-1 alpha Quantikine ELISA Kit were from R&D Systems. Alexa Fluor® fluorescent dye-conjugated secondary antibodies were from Life Technologies for immunofluorescent staining.
Shen K., Luk S., Hicks D.F., Elman J.S., Bohr S., Iwamoto Y., Murray R., Pena K., Wang F., Seker E., Weissleder R., Yarmush M.L., Toner M., Sgroi D, & Parekkadan B. (2014). Resolving Cancer-Stroma Interfacial Signaling and Interventions with Micropatterned Tumor-Stromal Assays. Nature communications, 5, 5662.
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6

Evaluating EMT-related Protein Expression

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Cells were lysed with the RIPA buffer containing 25 mM Tris-HCL, pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA and 1.0% NP-40 on ice. Samples were resolved by SDS-PAGE and transferred to PVDF membranes. The western blots were performed with antibodies against MLL3 (Polyclonal, Millipore Sigma, ABE1851, 1:1000), E-cadherin (clone 36, BD Biosciences, cat# 610182, 1:1000), Vimentin (clone D21H3, Cell Signaling Technology, cat# 5741S, 1:1000), Slug (clone C19G7, Cell Signaling Technology, cat#9585S, 1:1000), Snail (clone C15d3, Cell Signaling Technology, cat#3879S, 1:1000), Zeb1 (clone D80D3, Cell Signaling Technology, cat#3396S, 1:1000), Stat1 (clone D1K9Y, Cell Signaling Technology, cat#14994S, 1:1000), phosphor-Stat1(Tyr701) (clone 58D6, Cell Signaling Technology, cat#9167S,1:1000), β-actin (clone C4/actin, BD Biosciences, cat#612656, 1:10,000), H3K27ac (clone D5E4, Cell Signaling Technology, cat#8173S, 1:1000), H3K4me1 (clone D1A9, Cell Signaling Technology, cat#5326S, 1:1000), H3K4me3 (clone C42D8, Cell Signaling Technology, cat#9751S, 1:1000), or Histone H3 (clone 1B1B2, Cell Signaling Technology, cat#14269S, 1:10,000). Original scans of the western blot results are provided in the Source Data for individual figures.
Cui J., Zhang C., Lee J.E., Bartholdy B.A., Yang D., Liu Y., Erler P., Galbo PM J.r., Hodge D.Q., Huangfu D., Zheng D., Ge K, & Guo W. (2023). MLL3 Loss Drives Metastasis by Promoting a Hybrid Epithelial-Mesenchymal Transition State. Nature cell biology, 25(1), 145-158.
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