Protein extraction and western blotting analysis were performed using previously standard procedures (17 (link)). The following antibodies were used for western blotting: anti-E-cadherin antibody(#20874-1-AP Proteintech, China), anti-N-cadherin antibody(#22018-1-AP Proteintech, China), anti-ZEB1 antibody(#66279-1-Ig Proteintech, China), anti-Vimentin antibody(10366-1-AP Proteintech, China), anti-SOX2 antibody(#11064-1-AP Proteintech, China), anti-GAPDH antibody(#10494-1-AP Proteintech, China), and anti-PD-L1 antibody(#66248-1-Ig Proteintech, China).
Anti vimentin antibody
Manufactured by Proteintech
Sourced in United States, China
The Anti-vimentin antibody is a laboratory reagent used for the detection and identification of the vimentin protein. Vimentin is a type III intermediate filament that is expressed in various cell types, particularly mesenchymal cells. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to visualize and analyze the presence and distribution of vimentin in biological samples.
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Lab products found in correlation
Anti e cadherin antibody, by Proteintech (5 mentions)
Anti n cadherin antibody, by Proteintech (4 mentions)
Anti gapdh antibody, by Proteintech (3 mentions)
Bx51 light microscope, by Olympus (3 mentions)
Anti e cadherin antibody, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti zeb1 antibody, by Proteintech (1 mentions)
Anti sox2 antibody, by Proteintech (1 mentions)
Protein a g agarose, by Beyotime (1 mentions)
Anti sirt1 antibody, by Cell Signaling Technology (1 mentions)
Protein a g magnetic beads, by Selleck Chemicals (1 mentions)
Ip lysis buffer, by Bio-Rad (1 mentions)
Laemmli protein sample buffer, by Bio-Rad (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd31 antibody, by Abcam (1 mentions)
Goat anti rabbit igg h l horseradish peroxidase, by Beyotime (1 mentions)
Anti zo 1 antibody, by Proteintech (1 mentions)
Anti mmp2 antibody, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti c myc antibody, by Abcam (1 mentions)
16 protocols using anti vimentin antibody
1
Protein Extraction and Western Blotting
2
Immunoprecipitation of MXRA7 and Vimentin
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NIH3T3 cells were harvested into Co-IP buffer (Tris-HCl 50mM, pH7.4, MgCl2 10mM, NaCl 150mM, TritonX-100, 0.5%) supplemented with protease inhibitors and centrifuged at 12,000 g for 15 min. The supernatant was mixed with protein A/G agarose (Beyotime Biotechnology, Shanghai, China) at 4°C for 30 min to remove nonspecific binding constituents. Eight hundred micrograms of recovered proteins was mixed with 1μg anti-MXRA7 antibody (SZ181) or anti-vimentin antibody or isotype control IgG (Beyotime, Shanghai, China) at 4°C overnight on a roller. Then, 40μL protein A/G agarose beads were added for another 2 h at 4°C. The beads were spin-washed with pre-cooled Co-IP buffer six times. The final sediments were resuspended in an SDS loading buffer and boiled for 10 min. Cleared samples were separated on SDS-PAGE followed by transferring to the NC membrane. Routine WB was performed with an anti-MXRA7 antibody (1:1000 dilution, Sigma-Aldrich Company, St Louis, MO) or anti-vimentin antibody (1:1000 dilution, Proteintech, Wuhan, China).
3
Immunoprecipitation of SIRT1 and Vimentin
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Anti‐SIRT1 antibody (Cell Signaling Technology, #8469), anti‐vimentin antibody (Proteintech, #10366‐1), or matched IgG isotype antibody (Cell Signaling Technology, #5946) were incubated with Protein A/G Magnetic Beads (Bimake, Houston, USA, #B23202) for 1.5 h at room temperature. Cells and tissues were lysed with IP lysis buffer (Thermo Fisher Scientific, Waltham, USA, #87787), and a total of 400 μg of each sample lysate was incubated with an antibody−bead complex for another 8 h. Then, the protein extracts were precipitated by the antibody−bead complex using a magnetic rack. After being washed 3 times with IP lysis buffer, Co‐IP products were boiled at 95°C for 15 min with diluted 4 × Laemmli protein sample buffer (Bio‐Rad, Hercules, USA, #1610747) in lysis buffer. Finally, proteins were resolved by SDS/PAGE and immunoblotted with antibodies as indicated.
4
Histological Analysis of Lung Tissues
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Para n-embedded lung tissues were sectioned at 5 μm thick, then stained with hematoxylin and eosin (H&E). Pulmonary tissues were cut into 4 μm sections, then depara nized and rehydrated. For retrieving antigen, the slides were heated at 95 °C in 0.01 M citrate buffer (pH = 6.0), and 3% hydrogen peroxide was used to quench peroxidase activity for 20 min. The sections were treated with normal goat serum, followed by incubation overnight with anti-Vimentin antibody (1:400 dilution; Proteintech) at 4 °C. After being rinsed with PBS, the sections were incubated with goat anti rabbit IgG for 1 h and stained with 3, 3′diaminobenzidine (DAB; Zhongshan biotech, Beijing, China). After hematoxylin counterstain was completed, all the sections were dehydrated and sealed. We used PBS instead of prime antibody as a negative control. All images were recorded by Olympus BX-51 light microscope.
5
Immunohistochemical Analysis of Tumor Microenvironment
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Tumor-bearing mice were anesthetized with 1% pentobarbital and perfused with 4% paraformaldehyde. Tumors were captured and fixed in 4% paraformaldehyde, and paraffin sections of each group were serially sliced (6 μm thick) and subjected to immunohistochemical staining using the following antibodies: anti-CD31 antibody (Abcam, 1:500), anti-vimentin antibody (Proteintech, 1:100) and anti-E-cadherin antibody (Abcam, 1:500). The prepared slides were incubated overnight at 4°C and then incubated with goat anti-rabbit IgG (H+L) horseradish peroxidase (Beyotime Institute Biotechnology, Haimen, Jiangsu, People’s Republic of China; 1:100) for 40 min at 37°C. The antibody stainings were visualized with 3,3′-diaminobenzidine (DAB). A total of five areas were calculated per sample.
6
Comprehensive Protein Analysis via Western Blotting
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Total proteins from the cells were extracted with Laemmli buffer, incubated at 90°C for 5 min and centrifuged at 12,000× g for 20 min. Protein concentrations were measured using the BCA method. For the Western blot assay, proteins were separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gel (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA), blocked in 5% bovine serum albumin (BSA) with 0.1% Tween 20 in tris buffered saline (TBS) for 1 h at room temperature (RT) and, subsequently, incubated with the following primary antibodies: anti-vimentin antibody (Protein-tech, 1:3,000), anti-E-cadherin antibody (Abcam, 1:3,000), anti-ZO-1 antibody (Proteintech, 1:3,000), anti-MMP-2 antibody (Abcam, 1:3,000), anti-c-Myc antibody (Abcam, 1:3,000) and anti-GAPDH antibody (Proteintech, 1:3,000) overnight at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000, Thermo Fisher Scientific). The resultant bands were visualized using an enhanced chemiluminescence (ECL) detection system (ChemiDoc XRS; Bio-Rad, Hercules, CA, USA) and quantified using Quantity One software (Bio-Rad).
7
Evaluating TNBC Metastasis in Mice
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For in vivo metastasis analysis of TNBC cells, we performed tail vein injection of 1 × 106 MDA-MB-468 cells expressing either scramble or LIPG shRNA into athymic nude mice. Four weeks after injection, experimental mice were euthanized and dissected for lung isolation. Isolated lung were fixed and embedded in paraffin for making tissue sections. Paraffin-embedded lung tissue sections were stained with hematoxylin and eosin (HE staining) for histological analysis of metastatic tumor foci. IHC analysis of vimentin protein expression in lung tumor foci was also performed using the anti-vimentin antibody (Proteintech, Rosemont, IL) to confirm metastasis of MDA-MB-468. The numbers of lung tumor foci were counted according to HE and IHC staining of lung tissue samples. In vivo metastasis experiments were performed according to the animal protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland School of Medicine, which is in accordance with the guidelines established by the USPHS.
8
Immunohistochemical Analysis of Cancer Biomarkers
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Thirty-six representative samples from C1 (N = 18) and C2 (N = 18) were selected for tissue microarray (TMA) construction, the TMA is prepared as previously described43 . For immunohistochemistry staining, the sections were stained using anti-Vimentin antibody (Proteintech, 1:2000), anti-Ki67 antibody (Proteintech, 1:5000), anti-Cyclin D antibody (Proteintech, 1:1500), anti- E-cadherin antibody (Proteintech, 1:2000), anti- N-cadherin antibody (Proteintech, 1:200), anti-ADH1A antibody (Abcam, 1:500), anti-CYP3A4 antibody (Proteintech, 1:200), anti-CD34 antibody (Proteintech, 1:1000), anti-VEGFA antibody (Proteintech, 1:200), anti-CD8-alpha antibody (Abcam, 1:500), anti-CD163 antibody (Proteintech, 1:1000). Images were captured by MoticEasyScan (Motic).
9
Vimentin Protein Stability Assay
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To assess the stability of the Vimentin protein, cardiomyocytes were incubated with H2O2 and treated with 7.50 μg/mL protein synthesis inhibitor cycloheximide (Selleckchem, Houston, TX) for the indicated time periods or 1 μM proteasome inhibitor MG132 (MedChemExpress, NJ) for 6 h before harvest. Vimentin was immunoblotted with an anti-Vimentin antibody (Proteintech) and β-actin (Proteintech) was considered as a reference.
10
Quantitative Protein Analysis by Western Blot
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Total cellular proteins were lysed by RIPA buffer containing protease inhibitors (Sigma, USA). The protein extracts were harvested and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extractions were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After incubation with a high-affinity anti-KRT6B antibody (1:2000, Proteintech, USA), anti-vimentin antibody (1:1000, Proteintech, USA) or anti-GAPDH antibody (1:1000, Cell Signaling Technology, USA), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). After washing, the signals were detected using a chemiluminescence system (Millipore, USA) and analysed using Image Lab Software (Bio-Rad, USA).
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