Western blot assay was performed using the standard method as briefly described 21 (link),22 (link). Protein samples were collected after cells were lysed and quantified. 20~30 μg/lane protein were separated by 10%~15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A wet transfer (Bio-Rad, USA) were then performed to transfer the protein onto polyvinylidene fluoride (PVDF) membranes and blocked with non-fat milk for 1 h. The membranes were incubated with primary antibody MFAP5 (HPA010553,Sigma,Germany), MMP9 (D6O3H, Cell signaling technology, USA), MMP2 (D4M2N Cell signaling technology, USA), vimentin (D21H3, Cell signaling technology, USA), Sanil (C15D3, Cell signaling technology, USA), AKT (11E7, Cell signaling technology, USA), p-AKT (D9E, Cell signaling technology, USA), HIF-1α (D1S7W, Cell signaling technology, USA), α-tubulin (11224, Proteintech, USA), β-actin (20536 Proteintech, USA) at 1:1000 dilution overnight and then with anti-rabbit HRP-linked IgG secondary antibody(7074, Cell signaling technology, USA) at 1:2000 dilution for 1 hour. The immunoreactive bands were visualized with ECL Ultra (New Cell and Molecular Biotech, Suzhou, China). α-tubulin and β-actin was used as an internal control.
Vimentin d21h3
Manufactured by Cell Signaling Technology
Sourced in United States
Vimentin (D21H3) is a rabbit monoclonal antibody that recognizes the vimentin protein. Vimentin is a Type III intermediate filament protein that is expressed in mesenchymal cells. This antibody can be used for the detection of vimentin in various applications such as immunohistochemistry and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin 24e10, by Cell Signaling Technology (6 mentions)
Mem vitamin solution, by Thermo Fisher Scientific (2 mentions)
Necrox 5, by Santa Cruz Biotechnology (2 mentions)
Antibodies to tnfα and mekk4, by Abcam (2 mentions)
Noxa antibody, by Novus Biologicals (2 mentions)
Rack1 antibody, by BD (2 mentions)
Cell culture grade amino acids, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
E cadherin 4a2, by Cell Signaling Technology (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Wet transfer, by Bio-Rad (1 mentions)
Mmp9 d6o3h, by Cell Signaling Technology (1 mentions)
Hrp linked anti rabbit igg secondary antibody, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
α tubulin, by Proteintech (1 mentions)
Ab5694, by Abcam (1 mentions)
Ecl antirabbit igg horseradish peroxidase linked whole antibody, by GE Healthcare (1 mentions)
25 protocols using vimentin d21h3
1
Western Blot Protocol for Protein Analysis
2
Immunofluorescence Staining of Paraffin Sections
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Paraffin sections at a thickness of 4 µm were used for immunofluorescence staining. The primary antibodies were against α-SMA (ab5694; Abcam, Cambridge, UK), vimentin (D21H3, #5741, Cell Signaling, Beverly, MA, USA) and fibronectin (F3647, Sigma-Aldrich, St. Louis, MO, USA). The secondary antibodies were Alexa Fluor® 488-conjugated antibodies (Jackson Immuno-Research Laboratories, West Grove, PA, USA). For nuclei staining, 4,6-diamidino-2-phenylindole (DAPI) was used. All sections were visualized under a confocal microscope (LSM880, Carl Zeiss, Oberkochen, Germany). The interstitial areas of α-SMA, vimentin, and fibronectin on immunostaining were quantified in 10 regions of randomly selected fields using Image J software, and the results were expressed as a percentage of the cortical area stained (large blood vessels were excluded from the analysis for α-SMA staining).
3
Western Blot Antibody Validation
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HSP27 (G31) mouse monoclonal antibody and GAPDH (14C10), E-cadherin (24E10), and vimentin (D21H3) rabbit monoclonal antibodies were obtained from Cell Signaling Technology (Danvers, MA). ECL Anti-Rabbit IgG, Horseradish Peroxidase Linked whole antibody and ECL Anti-Mouse IgG, Horseradish Peroxidase Linked whole antibody were obtained from GE Healthcare UK (Buckinghamshire, UK)
4
Western Blot Analysis of Protein Markers
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Protein extracts from cells were harvested and immunoblotted32 (link). The following antibodies were used for immunoblotting: Notch1 (Val 1744; D3β8), BMI1 (D20B7), Snail (C15D3), Vimentin (D21H3), and E-cadherin (24E10) were from Cell Signaling Technology. Twist1 (3E11) was from Abnova and β-actin (BA3R) was from Thermo Scientific. Enhanced Chemiluminescence Substrate (PerkinElmer) and Gene GNOME (Syngene) was used for visualization. Chemiluminescence signals were quantitated using NIH Image J (National Institutes of Health).
5
Protein Extraction and Western Blot Analysis
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Cell monolayers were lysed in lysing buffer containing 1M Tris-HCl (pH 7.5), 3M NaCl, NP4O, distilled water, a protease inhibitor cocktail (Roche, Switzerland), PMSF and sodium orthovanadate. Cells were centrifuged at 13 000 RPM, 4°C. The amount of protein was measured using the Bradford assay [20 (link)] and stored at -80°C until used. Equal amounts of protein (20 μg) were run on Bolt® Bis-Tris Plus gels (Invitrogen, Thermo Fisher Scientific) and transferred to a nitrocellulose membrane using iBlot® Dry Blotting System (Invitrogen, Thermo Fisher Scientific). The membranes were blocked with 5% skim milk in a TBS buffer with 1% of Tween 20 for 1h and incubated with primary antibodies against vimentin (D21H3) (Cell Signaling Technology, MA, USA) and β1 integrin (Cell Signaling Technology, MA, USA) at 4°C overnight. Membranes were washed 3 times in TBS and incubated with suitable secondary antibodies and then washed 3 times in TBS. Signals were detected using LumiGLO® chemiluminescent substrate (Cell Signalling Technology, Danveers, MA).
6
Immunohistochemical Protein Expression Analysis
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The sections were heated in a microwave oven in citrate buffer (pH =6.0) for 20 minutes at 95°C and cooled at room temperature. The sections were then washed with phosphate buffered saline (PBS) for three times (3 minutes/time). Endogenous peroxidase activity was blocked in 3% H2O2 for 20 minutes at room temperature. After washing with PBS for three times (3 minutes/time), sections were incubated with normal goat serum for 30 minutes at room temperature. The sections were then incubated overnight at 4°C with specificity primary antibody (anti-ERα [ab37438], Abcam, UK; anti-ERβ [ab3576], Abcam; PI3 kinase p110α [C73F8], Cell Signaling, USA; AKT [C73H10], Cell Signaling; E-cadherin [24E10], Cell Signaling; vimentin [D21H3], Cell Signaling) 37°C rewarming for 45 minutes. The sections were washed three times with PBS (3 minutes/time), then incubated with GTVision (Gene Technology Shanghai Co., Ltd., Shanghai, People’s Republic of China) for 30 minutes at 37°C. The sections were then washed three times with PBS and visualized with diaminnobenzidinetetra-hydrochloride (DAB kit; Shanghai Secco Chemical Technology Co., Ltd., People’s Republic of China). Finally, the sections were counterstained with hematoxylin and dehydrated.
7
Immunofluorescent Labeling of Vimentin and Keratin 7
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Cells were fixed in 4% paraformaldehyde and blocked in 0.3% Triton X-100 and 1% bovine serum albumin, and then incubated with primary antibodies against vimentin (D21H3) and keratin 7 (D1E4) (Cell Signaling Technology, Danvers, MA, USA). Appropriate second antibodies conjugated with Alexa fluorochromes were used to detect positive staining. The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI), and positively stained cells were visualised under a fluorescence microscope.
8
Protein Expression Analysis by Western Blot
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Cell lysate, SDS-polyacrylamide gel electrophoresis, and Western blots were performed as previously described24 (link). The sources of antibodies were as indicated: CD147 (ab666; Abcam, Cambridge, UK) and HSP70/HSC73 (1B5; ENZO Life Science, Plymouth Meeting, PA, USA). The following antibodies were from Cell Signaling Technology (Danvers, MA, USA): vimentin (D21H3), N-cadherin (D4R1H) XP, claudin-1 (D5H1D) XP, Slug (C19G7), E-cadherin (24E10), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, and HRP-conjugated anti-mouse IgG.
9
Western Blot Analysis of Cell Signaling
10
Western Blot Analysis of EMT Markers
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The cells were lysed using RIPA buffer containing phosphatase inhibitor cocktail I (Sigma) and a protease inhibitor cocktail mini-tablet (Roche Diagnostics, Indianapolis, IN). The proteins in the lysates (20 μg) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After the membrane was blocked with 5% non-fat milk in TBST, it was incubated with the following primary antibodies over night at 4°C : E-cadherin (ab76055, 1:1,000; Abcam, Cambridge, MA, USA), vimentin (D21H3, 1:1,000; Cell Signaling Technology, Danvers, MA, USA), ZEB1 (NBP2-23484; 1:800, Novus Biologicals, Littleton, CO, USA), Twist1 (ab175430, 1:1000, Abcam), Snail1(C15D3, 1:1,000; Cell Signaling Technology, Danvers, MA, USA), a-SMA (ab5694, 1:1,000; Abcam, Cambridge, MA, USA) and GAPDH (14C10, 1:1,000; Cell Signaling Technology). The membrane was then washed three times with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit and goat anti-mouse (1:5,000) immunoglobulin G (Invitrogen). The western blots were visualized using the enhanced chemiluminescence reagents (Millipore, WBKLS0100), and the western blotting results were semi-quantified using ImageJ software (NIH, USA).
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