Anti human vimentin

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-human vimentin is a primary antibody that specifically recognizes the vimentin protein, which is a type III intermediate filament protein expressed in various cell types. It is commonly used in immunohistochemistry and Western blotting applications to detect and quantify vimentin expression in samples.

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7 protocols using anti human vimentin

Western Blot Analysis of EMT and Cytokine Markers

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Cell lysate was prepared in radioimmunoprecipitation assay buffer lysis buffer
and debris completely removed by refrigerated centrifugation. The protein
concentration was determined by the bicinchoninic acid Protein Assay Kit
(ThermoFisher, MA, USA). An equal amount of protein was resolved by SDS-PAGE and
then transferred onto polyvinylidene difluoride (PVDF) membrane on ice. After
brief blocking with 5% skim milk in Tris-buffered saline with 0.1% Tween-20
(TBST), the PVDF membrane was hybridized with indicated primary Abs
(anti-human-E-cadherin, 1:1000, #14472; anti-human-N-cadherin, 1:1000, #14215;
anti-human-vimentin, 1:1000, #3390; anti-mouse-IL-6, 1:1000, #12912;
anti-mouse-TNF-α, 1:1000, #11948; anti-GAPDH, 1:1000, #2118; Cell Signaling
Technology, MA, USA) at 4°C overnight. The unbound Abs were washed off with TBST
next day and incubated with specific secondary Abs (anti-rabbit IgG, HRP-linked
Ab, 1:5000, #7074; anti-mouse IgG, HRP-linked Ab, 1:5000, #7076; Cell Signaling
Technology, MA, USA) at room temperature (25°C) for 1 h. The PVDF membrane was
then rigorously washed with TBST for 30 min and target protein bands were
visualized using the Enhanced Chemiluminescence Kit (Millipore, UT, USA) in
accordance with the manufacturer’s instructions. The endogenous GAPDH was
employed as loading control.

Wan G., Xie M., Yu H, & Chen H. (2018). Intestinal dysbacteriosis activates tumor-associated macrophages to promote epithelial-mesenchymal transition of colorectal cancer. Innate Immunity, 24(8), 480-489.

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Immunofluorescent Analysis of NSCLC Cells

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NSCLC cells were seeded into 48-well plates at a density of 3 × 10³ cells/well. Cells were fixed with 4% formaldehyde for 15 min, washed with PBS, treated with 5% bovine serum albumin (BSA) for 30 min at room temperature, and incubated with anti-E-cadherin (1:200) or anti-human vimentin (1:200) primary antibodies (Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. The cells were incubated with a FITC-conjugated anti-rabbit secondary antibody (Abcam, Cambridge, USA) at 4°C for 2 h. Nuclear staining was performed with DAPI (Sigma, St. Louis, MO, USA) at room temperature for 5 min. Following two washes with PBS, cells were observed using an inverted fluorescence microscope (Olympus, Tokyo, Japan).

Chen D., Xie S., Wu Y., Cui Y., Cai Y., Lan L., Yang H., Chen J, & Chen W. (2021). Reduction of Bladder Cancer Chemosensitivity Induced by the Effect of HOXA-AS3 as a ceRNA for miR-455-5p That Upregulates Notch1. Frontiers in Oncology, 10, 572672.

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Western Blot Analysis of EMT Markers

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The KFs with different treatment were harvested by centrifugation and washed with PBS. Cells were lysed in RIPA buffer containing protease inhibitors. The lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to Polyvinylidene Fluoride (PVDF) membranes by electroblotting. The membranes were incubated overnight at 4 °C with the primary antibodies after blocking with 5% nonfat milk. The anti-human E-cadherin, anti-human vimentin, anti-human PKM2, anti-human HIF-1α, and anti-human phospho-p70s6k primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The blots were then incubated with the secondary antibody for 2 h at room temperature. Each experiment was repeated for 3 times. Specific proteins were visualized using the ECL system (GE Healthcare) and the FUJIFILM Luminescent Image Analyzer LAS3000 (Fuji Film).

Lei R., Zhang S., Wang Y., Dai S., Sun J, & Zhu C. (2019). Metformin Inhibits Epithelial-to-Mesenchymal Transition of Keloid Fibroblasts via the HIF-1α/PKM2 Signaling Pathway. International Journal of Medical Sciences, 16(7), 960-966.

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Western Blot Analysis of EMT Markers

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The cells were harvested by centrifugation and washed with PBS. The cells were lysed in RIPA buffer containing protease inhibitors. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to Polyvinylidene Fluoride (PVDF) membranes by electroblotting. The membranes were blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween 20 for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies. The anti-human E-cadherin, anti-human vimentin, anti-human SNAILl2, anti-human STAT3, and anti-human phospho-p70s6k primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-human β-actin primary antibodies and Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse secondary antibodies were purchased from ZSGB-BIO (Beijing, China). After incubation with the secondary antibody for 1 h at room temperature, the protein bands were detected using the ECL detection system (BD Biosciences, New York, NY, USA).

Cheng K, & Hao M. (2016). Metformin Inhibits TGF-β1-Induced Epithelial-to-Mesenchymal Transition via PKM2 Relative-mTOR/p70s6k Signaling Pathway in Cervical Carcinoma Cells. International Journal of Molecular Sciences, 17(12), 2000.

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Protein Expression Analysis in Cells

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Total protein from cells was lysed using M-PER Mammalian Protein Extraction Reagent (Thermo) supplemented with a protease inhibitor cocktail (Sigma, St Louis, MO, USA). Samples were denatured, and equal amounts of protein were subjected to SDS-PAGE, and then transferred to nitrocellulose membrane. After blocking with 5% non-fat milk in TBST for 60 min, membranes were incubated with primary antibody dissolved in 5% bovine serum albumin in TBST overnight at 4°C. The following primary antibodies were used: anti-human- E-cadherin (1:2000, 24E10; Cell Signaling Technology, Danvers, MA, USA), anti-human-Vimentin (1:2000, D21H3; Cell Signaling Technology), anti-human-Snail (1:1000, C15D3; Cell Signaling Technology), anti-human-Twist (1:1000; Cell Signaling Technology), anti-human-Zeb (1:1000, D80D3; Cell Signaling Technology), and anti-human-Slug (1:1000, C19G7; Cell Signaling). Human GAPDH (1:5000; KangChen, Shanghai, China) was used as an internal reference.

Han Y., Ye J., Wu D., Wu P., Chen Z., Chen J., Gao S, & Huang J. (2014). LEIGC long non-coding RNA acts as a tumor suppressor in gastric carcinoma by inhibiting the epithelial-to-mesenchymal transition. BMC Cancer, 14, 932.

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Immunofluorescence Assay for Epithelial-Mesenchymal Transition

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HCC cells were seeded into 48-well plates at a density of 5×10³ cells/well. Cells were fixed with 4% formaldehyde for 15 min at 37°C, washed with PBS, blocked with 5% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) for 30 min at room temperature, and incubated with anti-E-cadherin (1:200, 3195) or anti-human vimentin (1:200, 5741) primary antibodies (Cell Signaling Technology, Inc.) at 4°C overnight. The cells were then incubated with foat anti-rabbit immunoglobulin G H&L (Alexa Fluor^® 488) antibodies (Abcam; 1:1000, ab150077) at 4°C for 2 h. Nuclear staining was performed with DAPI (Sigma-Aldrich; Merck KGaA) at room temperature for 2 min. Following washing with PBS three times, cells were observed using an inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Zhang Y., Lu Y., Zhang C., Huang D., Wu W., Zhang Y., Shen J., Cai Y., Chen W, & Yao W. (2018). FSCN-1 increases doxorubicin resistance in hepatocellular carcinoma through promotion of epithelial-mesenchymal transition. International Journal of Oncology, 52(5), 1455-1464.

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Western Blot Analysis of Protein Expression

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Protein lysates (50 µg per lane) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies (1:500 dilution) at 4°C overnight, followed by horseradish peroxidase-conjugated secondary antibodies (1:3,000 dilution). Anti-human RBM8A (Santa Cruz, sc-32312), anti-human actin (Proteintech, HRP-60008), anti-human snail (Cell Signaling Technology, 3879), anti-human p-stat3 (Cell Signaling Technology, 9131), anti-human stat3 (Cell Signaling Technology, 12640), anti-human fibronectin (Sigma-Aldrich, F3648), anti-human vimentin (Cell Signaling Technology, 5741), anti-human Notch (Abways Technology, CY52444) and donkey anti-rabbit IgG (Cell Signaling Technology, 7074) were used in this study. Immune-reactive proteins were visualized by enhanced chemiluminescence (ECL).

Ni X., Cao X., Wu Y, & Wu J. (2017). FSTL1 suppresses tumor cell proliferation, invasion and survival in non-small cell lung cancer. Oncology Reports, 39(1), 13-20.

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