Cells grown on glass coverslips were fixed in 4% formaldehyde in PBS at room temperature for 15 min and permeabilized with 0.1% Triton X‐100 in PBS at room temperature for 5 min. To detect endogenous vimentin in SW‐13 cells, we incubated the cells overnight at 4 °C with rabbit anti‐vimentin antibody (1 : 100 dilution) (Cell Signaling Technology, Danvers, MA, USA). To detect endogenous cytokeratin 8 in MCF‐7 cells, we incubated the cells overnight at 4 °C with rabbit anti‐(cytokeratin 8) antibody (1 : 200 dilution) (Abcam, Cambridge, UK). After washing three times with PBS, the cells were incubated for 1 h with Alexa Fluor 405‐conjugated goat anti‐rabbit IgG antibody (1 : 200 dilution) (Abcam). When required, cell nuclei were stained with 1 µg·mL−1 4', 6‐diamidino‐2‐phenylindole (DAPI) (Dojindo, Kumamoto, Japan) for 5 min at room temperature. Fluorescently labeled cells were examined using an Olympus BX53 fluorescence microscope (Olympus, Tokyo, Japan) and a Nikon C1 confocal microscope (Nikon, Tokyo, Japan).
Rabbit anti vimentin antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-vimentin antibody is a primary antibody that recognizes the vimentin protein, which is a type III intermediate filament found in various cell types. This antibody can be used for the detection and localization of vimentin in research applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Rabbit anti cytokeratin 8 antibody, by Abcam (1 mentions)
Bx53 fluorescence microscope, by Olympus (1 mentions)
C1 confocal microscope, by Nikon (1 mentions)
A1r point scanning confocal microscope, by Nikon (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Mouse anti tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse anti lamin b antibody, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Mouse anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl western blot detection reagents, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Lc3b antibody, by Merck Group (1 mentions)
Ampk alpha 1 monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Mtor monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Il 37 polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
Application suite software, by Leica (1 mentions)
Dm6000b microscope, by Leica (1 mentions)
6 protocols using rabbit anti vimentin antibody
1
Immunofluorescence Staining of Cytoskeletal Proteins
2
Quantifying Fibroblast Infiltration in Integra Grafts
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Confocal microscopy was performed as described previously with minor modifications [43 (link)]. Briefly, cryopreserved graft sections (n = 4–6 mice per group) were permeabilised with methanol (10 min), washed, blocked (30 min), and incubated with rabbit anti-vimentin antibody (1:600, Cell Signalling, Danvers, MA, USA) overnight. Sections were washed and probed with donkey anti-rabbit Ig–AF647 antibody (Invitrogen, Carlsbad, CA, USA) for 60 min and washed excessively prior to mounting using Prolong Gold (Invitrogen, Carlsbad, CA, USA). For analysis, high-resolution images were acquired on Nikon A1R point scanning confocal microscope with Plan Fluo 20 x MIm/0.75 NA objective × 2 optical zoom. Six fields of view were randomly selected in specified areas of the graft. Integra® spectral profile in the 405–550 nm range interfered with fibroblast detection and counting. We, therefore, performed spectral unmixing of the stained Integra® graft images using NIS Analysis (Nikon, Japan), assigning all Integra® spectral property to the green channel (500–550nm). Evaluation of fibroblast infiltration was performed by thresholding vimentin-positive cells, counted with NIS Analysis software (Nikon, Japan).
3
Cloning and Reporter Constructs of LINC01010
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The full-length of LINC01010 or LINC01010-S1-tagged (S1 sequence 5′-ACCGACC AGAATCATGCAAGTGCGTAAGATAGTCGCGGGCCGGG-3′, an RNA aptamer which binds the streptavidin) genes were cloned into the lentiviral vector pLentilox3.7 to generate pll3.7-LINC01010 and pll3.7-LINC01010-S1, respectively. The mCherry-Vimentin-7 was a gift from Michael Davidson (Addgene plasmid#55156) [53 (link)]. The promoter region from –952 to –16 nt in the human LINC01010 gene was subcloned into the pGL2-basic vector to construct a reporter named pGL2-LINC01010-luc. The following antibodies were purchased from the indicated companies: rabbit anti-vimentin antibody (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Phospho-vimentin (Ser56) antibody (Affinity Biosciences, Cincinnati, OH, USA), rabbit anti-Phospho-vimentin (Ser83) antibody (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Phospho-vimentin (Ser72) antibody (ZEN-BIOSCIENCE, Chengdu, China), mouse anti-GAPDH antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-lamin B antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), horseradish peroxidase HRP-conjugated secondary antibodies (The Jackson Laboratory, Bar Harbor, ME, USA).
4
Immunohistochemical Analysis of Cytokeratin and Vimentin
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A volume fraction of 95% alcohol was purchased from Shandong Anjie Gaoke Technology Co., Ltd. (Shandong, China). The GH was purchased from Anhui Anke Bio-Tech Co., Ltd. (Hefei, Anhui, China). The rabbit anti-cytokeratin antibody was purchased from Novus Biologicals (Littleton, CO, USA), and the rabbit anti-vimentin antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The immunohistochemistry (IHC) kit was purchased from Beijing Zhongshang Jinqiao Co., Ltd. (Beijing, China) and the microscope was purchased from Olympus Corp. (Shinjuku, Tokyo, Japan).
5
Western Blot Analysis of SKOV3 Cells
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SKOV3 FHC-silenced and non-silenced cells were lysed in the following buffer [20 mM Hepes pH 7.9, 420 mM NaCl, 1% Triton X-100, 1 mM EDTA, 25% glycerol, 1 mM PMSF, 1 mM Na3VO4, 1 mM DTT, 1 μg/ml aprotinin, 1 μg/ml leupeptin] for 30 min on ice. After removing cell debris by centrifugation (12,000g × 30 min), the concentration of protein extracts was measured by the Bio-Rad protein assay according to the manufacturer's instructions (BioRad Laboratories). A total of 70 μg protein extract was boiled for 10 min in SDS sample buffer, separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane by electroblotting. Non-specific reactivity was blocked in non fat dry milk in TPBS [5% (w/v) milk in PBS (pH 7.4) and 0.005% Tween 20] for 2 h at room temperature. The membrane was incubated with rabbit anti-H ferritin antibody (1:200; Santa Cruz Biotechnology, Texas, USA), rabbit anti-Vimentin antibody (1:500; Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-TCF8/ZEB1 antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA) over-night at 4°C, followed by incubation with goat anti-rabbit secondary antibody (1:5000; Santa Cruz Biotechnology). The membrane was developed by ECL-Western blot detection reagents according to the manufacturer›s instructions (Santa Cruz Biotechnology). γ-Tubulin was used as a loading control.
6
Immunohistochemistry for Dermal Markers
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Sections (5 mm) were stained with H&E to assess the general morphology. Paraffin sections were immunostained with lineage-specific antibodies to identify eosinophils and dermal fibroblasts by immunohistofluorescence. Samples were incubated with rat anti-mouse major basic protein (MBP) antibody (specific for eosinophils, a gift from James J. Lee, Ph.D., Mayo Clinic, Scottsdale, AZ, United States), rabbit anti-Vimentin antibody (specific for dermal fibroblasts, Cell Signaling Technology, Beverly, MA, United States), AMPK alpha-1 monoclonal antibody, mTOR monoclonal antibody, and IL-37 polyclonal antibody (Thermo Fisher Scientific, Rockford, IL, United States), and LC3B antibody (Sigma-Aldrich, St. Louis, MO, United States). Cy3-conjugated goat anti-rat immunoglobulin G (IgG) antibody (Beyotime Co., Shanghai, China) and Alexa Fluor 488-conjugated goat anti-rabbit and goat anti-mouse IgG antibody (ABclonal, MA, United States) were used as secondary antibodies. All images were acquired with a Leica DM6000B microscope (Leica Microsystems GmbH, Wetzlar, Germany) and processed using the Leica Application Suite software (Leica Microsystems GmbH).
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