Equal amounts of proteins extracted from the different groups were run on 10% SDS-PAGE and transferred to PVDF membranes (Amersham, St. Albans, UK). Membranes were incubated with mouse anti-vimentin monoclonal antibody (Santa Cruz; 1:500) and mouse anti-CK-19 monoclonal antibody (Santa Cruz; 1:500) respectively overnight at 4 °C. The membranes were then incubated with HRP-conjugated goat anti-mouse IgG (ZSJQ-Bio; 1:2000) at room temperature for 1 h. ECL detection reagents (Millipore) were added to the membranes and they were immediately exposed to x-ray film (Kodak, Rochester, NY, USA).The β-actin signal was used as a control to ensure equal protein loading.
Mouse anti vimentin monoclonal antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Mouse anti-vimentin monoclonal antibody is a research-grade tool used for the detection and analysis of vimentin, an intermediate filament protein expressed in various cell types. This antibody can be utilized in techniques such as immunohistochemistry, Western blotting, and flow cytometry to identify and study vimentin-containing cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Cytiva (1 mentions)
Ecl detection reagent, by Merck Group (1 mentions)
X ray film, by Kodak (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Trichostatin a, by Merck Group (1 mentions)
Anti e cadherin rabbit monoclonal antibody, by Cell Signaling Technology (1 mentions)
Nitroblue tetrazolium, by Promega (1 mentions)
Bsa pbs, by Santa Cruz Biotechnology (1 mentions)
Optical microscope, by Olympus (1 mentions)
Mouse monoclonal anti cd68 antibody, by Abcam (1 mentions)
Rabbit anti fap polyclonal antibody, by Abnova (1 mentions)
Mab1281, by Merck Group (1 mentions)
Mouse anti ki 67 monoclonal antibody, by Abcam (1 mentions)
Nis elements d v 4, by Nikon (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Rabbit monoclonal anti gfp antibody, by Cell Signaling Technology (1 mentions)
Oil red o, by Merck Group (1 mentions)
Rabbit polyclonal anti ki67 antibody, by Abcam (1 mentions)
Oil red o, by Santa Cruz Biotechnology (1 mentions)
9 protocols using mouse anti vimentin monoclonal antibody
1
Western Blot Analysis of Vimentin and CK-19
2
Immunofluorescence Analysis of Paraffin-Embedded Tissues
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Paraffin-embedded tumor tissues were cut in 4-μm sections, deparaffinized in xylene, and rehydrated through a graded ethanol series. Next, the sections were rinsed three times in 1X PBST (PBS with Tween 20). The sections were then blocked in 1X PBS containing 0.5% Triton X-100 and 5% sheep serum for 30 min at room temperature before incubating them overnight at 4 °C with a rabbit anti-Prx II polyclonal antibody (AbFrontier technology, Seoul, South Korea) and a mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology). Sections were again rinsed three times in 1X PBST. To detect the bound primary antibodies, the sections were incubated in the dark at room temperature for 90 min with goat anti-rabbit and goat anti-mouse antibodies (Santa Cruz Biotechnology). Slides were counterstained for 20 min in the dark with DAPI diluted in 1X PBS before visualization and image capturing under a microscope.
3
Antibody-based protein analysis protocol
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The following primary antibodies were used in the present study: Rabbit anti-E-cadherin monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA; cat. no. 3195), rabbit anti-N-cadherin monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 4061P), rabbit anti-Slug monoclonal antibody (Cell Signaling Technology, Inc.; cat. no. 9585P), mouse anti-vimentin monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-15393), mouse anti-SIP1 monoclonal antibody (E-11; Santa Cruz Biotechnology, Inc.; cat. no. sc-271984), rabbit anti-SIP1 monoclonal antibody (Abcam, Cambridge, UK; cat. no. ab-138222), mouse anti-HBx polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-57760), mouse anti-HDAC1 polyclonal antibody (Santa Cruz Biotechnology, Inc.; cat. no. sc-81598), rabbit anti-HDAC1 monoclonal antibody (GeneTex, Inc., Irvine, CA, USA; cat. no. GTX100513222) and mouse anti-β-actin monoclonal antibody (Boster Biological Technology, Ltd., Wuhan, China; cat. no. BM0627). The HDAC inhibitor trichostatin A (TSA) was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany; cat. no. T1952). DAPI (Beyotime Institute of Biotechnology) and Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) were used according to the manufacturer's protocols.
4
Western Blot Analysis of Protein Targets
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The samples were electrophoresed in 4–12% Bis-Tris gels at 100 V using MES running buffer. The proteins were electrophoretically transferred to a nitrocellulose membrane at 100 V for 1.5 h. The membrane was blocked with 3% milk in PBS 1X at 37°C for 2 h. The blots were incubated with primary antibodies: rabbit anti human LASP-1 polyclonal antibody, 1:500 in 0.3% BSA-PBS (Millipore); rabbit anti-green fluorescent protein polyclonal antibody, 1:100 in 0.3% BSA-PBS (Santa Cruz Biotechnology); mouse anti-vimentin monoclonal antibody, 1:1000 in 0.3% BSA-PBS (Santa Cruz Biotechnology) at room temperature overnight, washed three times with PBS, then incubated with an alkaline-phosphatase-conjugated secondary antibody (1:7500 in 0.3% BSA-PBS) for 4 h at 37°C. The results of the immunoreaction were detected with Nitroblue tetrazolium and bromochloroindolyl phosphate (Promega).
5
Immunohistochemical Analysis of Fibroblast Markers
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The BM tissue samples were used for immunohistochemistry and Gomori’s stain. The rabbit anti-FSP1/S100A4 monoclonal antibody (Millipore, Billerica, MA, USA), rabbit anti-FAP polyclonal antibody (Abnova, Taipei, Taiwan), goat anti-α-SMA polyclonal antibody (Abcam, Cambridge, MA, USA), rabbit anti-GDF15 monoclonal antibody (Cell Signaling, Danvers, MA, USA), mouse anti-Vimentin monoclonal antibody (Santa Cruz, Dallas, CA, USA), and mouse anti-CD68 monoclonal antibody (Abcam) incubations were performed according to the manufacturer’s instructions. Images of the slides stained for these markers were scanned at 40× magnification using the optical microscope (Olympus Co., Tokyo, Japan). The positive rate of each marker was quantified by counting the ratio of the positive cells in all nuclear cells. The reticulin fiber density (RFD) of the samples was assessed according to Norén-Nyström et al [18 (link)]. All readings and estimations were performed in a blinded manner.
6
Immunohistochemical and Immunofluorescence Analysis of Uterine Regeneration
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The expression of vimentin, Ki-67, and CD31 proteins was detected by immunohistochemistry. Slides were incubated with mouse anti-vimentin monoclonal antibody (Santa Cruz, CA, USA; 1:100), mouse anti-Ki-67 monoclonal antibody (Abcam, Cambridge, UK; 1:200), and mouse anti-CD31 monoclonal antibody (Santa Cruz; 1:100), respectively, followed by further incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZSJQ-Bio, Beijing, China, 1:100). The antibody stains were developed by the addition of diaminobenzidine (DAB) and the nucleus was stained with hematoxylin. Anti-human nuclear antibody MAB1281 (Millipore, Billerica, MA, USA) and TRITC-conjugated goat anti-mouse IgG (ZSJQ-Bio; 1:100) were used to detect the migration of the transplanted human UC-MSCs to the injured uterus by immunofluorescence; the nucleus was stained with DAPI.
7
Quantifying Vimentin Expression in Macrophages
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After COM treatment, the si-Control-transfected and si-Vimentin-transfected macrophages were adhered on a coverslip, fixed by 4% (v/v) paraformaldehyde/PBS at 25°C for 15 min, and then permeabilized with 0.2% Triton X-100/PBS at 25°C for 15 min. After washing, the cells were incubated at 4°C overnight with mouse monoclonal anti-vimentin antibody (Santa Cruz Biotechnology) (diluted 1:50 in 1% BSA/PBS). After washing, the cells were incubated with corresponding secondary antibody conjugated with Alexa Fluor 488 (Invitrogen) (diluted 1:2,000 in 1% BSA/PBS) at 25°C for 1 h. Finally, the cells were extensively washed with PBS and mounted onto a glass slide using 50% glycerol in PBS. The cells were imaged by using Nikon Eclipse 80i fluorescence microscope (Nikon). Expression level of vimentin was quantitated by measuring mean fluorescence intensity from at least 50 cells in 10 random HPF of each sample using NIS-Elements D V.4.11 software (Nikon).
8
Immunohistochemistry and Oil Red O Staining
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IHC staining was performed as previously described 16 (link) by using rat monoclonal anti-CD31 antibody (1:200, Santa Cruz), rabbit polyclonal anti-Ki67 antibody (1:200, Abcam), rabbit monoclonal anti-GFP antibody (1:300, Cell Signaling), mouse monoclonal anti-vimentin antibody (1:100, Santa Cruz), or mouse monoclonal anti-PPARγ2 antibody (1:200, Santa Cruz).
For Oil Red O staining, cells or 10 µm cryosections were fixed by neutral buffered 10% formalin, 30 min at room temperature. After rinsing in 60% isopropanol, cells were stained with Oil Red O (Sigma) at 60˚C for 5 min.
For Oil Red O staining, cells or 10 µm cryosections were fixed by neutral buffered 10% formalin, 30 min at room temperature. After rinsing in 60% isopropanol, cells were stained with Oil Red O (Sigma) at 60˚C for 5 min.
9
Antibody Characterization and Validation Protocol
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The following commercial antibodies were used at the dilution indicated: anti-hScrib goat polyclonal antibody (Santa Cruz WB 1:1000, IHC 1:100), anti-PP1γ goat polyclonal antibody (Santa Cruz WB 1:1000), anti-PP1 Gamma/PPP1CC Antibody LS-B4960 IHC-plus (tm) rabbit polyclonal antibody (Lifespan bioscience, Inc. IHC 1:200), anti-PP1γ sheep polyclonal antibody (Abcam, WB 1:1000), anti-actin monoclonal antibody (Sigma, WB 1:5000), mouse monoclonal anti-p53 (DO-1) (Santa Cruz WB 1:500), anti-p84 mouse monoclonal antibody (Abcam, WB 1:1000), anti-E-Cadherin rabbit polyclonal antibody (Santa Cruz WB 1:500), anti-α-tubulin mouse monoclonal antibody (Abcam, WB 1:1000), mouse monoclonal anti-vimentin antibody (Santa Cruz WB 1:500).
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