Anti vinculin fitc antibody

Manufactured by Merck Group

Sourced in United States, France

The Anti-vinculin-FITC antibody is a fluorescent labeled antibody that specifically binds to the protein vinculin. Vinculin is a cytoskeletal protein involved in cell-cell and cell-matrix adhesion. The FITC (Fluorescein isothiocyanate) label allows for the visualization and detection of vinculin in cellular and tissue samples.

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Lab products found in correlation

Rhodamine phalloidin rp, by Cytoskeleton (2 mentions) A1 n sim, by Nikon (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Lsm 780, by Zeiss (2 mentions) Thiazolyl blue tetrazolium bromide mtt, by Beyotime (1 mentions) Paraformaldehyde fix solution, by Beyotime (1 mentions) Actin tracker red rhodamine, by Beyotime (1 mentions) Live dead kit, by Beyotime (1 mentions) Bca protein assay kit, by Beyotime (1 mentions) Crystal violet staining solution, by Beyotime (1 mentions) Dimethyl sulfoxide (dmso), by Beyotime (1 mentions) 24 well transwell, by Corning (1 mentions) Complete alpha mem, by Merck Group (1 mentions) Duolink in situ reagents, by Merck Group (1 mentions) Tetramethylrhodamine b isothiocyanate tritc, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)

8 protocols using anti vinculin fitc antibody

Fabrication of Bioactive Scaffolds for Tissue Regeneration

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Nicotinic acid (NA), copper (II) acetate monohydrate, ethylene glycol were purchased from Aladdin Industrial Co. Ltd (Shanghai, China). Recombinant human basic fibroblast growth factor (bFGF) was purchased from Nanhai Longtime Pharmaceutical Co., Ltd. (Guang Dong, China). Gelatin methacryloyl (GelMA) was purchased from Engineering for Life (JiangSu, China). Chemicals for the preparation of Luria–Broth (LB) agar medium were purchased from Sangon Biotech Co. Ltd (Shanghai, China). BCA protein Assay kit, Live/Dead kit, Thiazolyl Blue Tetrazolium Bromide (MTT), 4% Paraformaldehyde Fix Solution, Dimethyl sulfoxide (DMSO), Crystal Violet Staining Solution, actin Tracker Red Rhodamine, DAPI were purchased from Beyotime (Shanghai, China). Phosphate buffered saline (PBS, pH 7.4), Fetal Bovine Serum (FBS), Dulbecco’s modified Eagle medium (DMEM), trypsin-EDTA and penicillin-streptomycin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-vinculin-FITC antibody was purchased from Sigma-Aldrich (St Louis, MO, USA). 24-well Transwell (8 μm pore size), Matrigel Matrix were purchased from Corning (New York, USA).

Wang T.L., Zhou Z.F., Liu J.F., Hou X.D., Zhou Z., Dai Y.L., Hou Z.Y., Chen F, & Zheng L.P. (2021). Donut-like MOFs of copper/nicotinic acid and composite hydrogels with superior bioactivity for rh-bFGF delivering and skin wound healing. Journal of Nanobiotechnology, 19, 275.

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Osteoblast Adhesion and Proliferation on Hydrogels

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Sterilized gels were punched into round disks using a cork borer (diameter 13.2 mm) and affixed to the bottom of 12-well TCPS cell culture plates coated with a layer of sterilized vacuum grease (Dow Corning, Midland, MI). MC3T3 pre-osteoblast cells were then seeded in complete alpha-MEM (Sigma) at a density of 30,000 cells cm⁻². The cell densities on hydrogels at 1, 4, 7, and 14 days post-seeding were determined by MTS assay. To observe the morphological features of adherent cells, gels were fixed after 7 days in culture with 4% paraformaldehyde (PFA) and further permeabilized through immersion in 0.2% Triton X-100 solution. After washing with PBS, cells were stained with anti-vinculin-FITC antibody (Sigma), rhodamine-phalloidin (RP, red), and 4’,6-diamidino-2-phenylindole (DAPI, blue) before being imaged on an inverted laser scanning confocal microscope (LSM 780, Carl Zeiss).

Liu X., George M.N., Li L., Gamble D., Miller AL I.I., Gaihre B., Waletzki B.E, & Lu L. (2020). Injectable Electrical Conductive and Phosphate Releasing Gel with Two-Dimensional Black Phosphorus and Carbon Nanotubes for Bone Tissue Engineering. ACS biomaterials science & engineering, 6(8), 4653-4665.

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Fluorescence Imaging of MC3T3 Cell Adhesion

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Fluorescence images of MC3T3 cells growing on the hydrogels were obtained at 4 days post-seeding. Cells were fixed with 4% paraformaldehyde (PFA) solution at room temperature for 10 minutes and permeabilized by 0.2% Triton X-100 solution at room temperature for another 10 min, prior to being immersed in 1% bovine serum albumin (BSA)/PBS solution at 37 °C for 30 minutes to block non-specific binding sites. Cells were then incubated with anti-vinculin-FITC antibody (1:50 in PBS, Sigma-Aldrich Co., Milwaukee, WI) for 1 hour to visualize cellular focal adhesion and with rhodamine-phalloidin (RP, 1:200 in PBS, Cytoskeleton Inc, Denver, CO) for 1 hour to stain the cell cytoskeleton. Cell nuclei were finally labeled by 4’,6-diamidino-2-phenylindole (DAPI) at 37 °C for 10 min. The stained MC3T3 cells were immediately imaged using the fluorescent microscope.

Xu H., Liu X., George M.N., Lee Miller A I.I., Park S., Xu H., Terzic A, & Lu L. (2021). Black phosphorus incorporation modulates nanocomposite hydrogel properties and subsequent MC3T3 cell attachment, proliferation, and differentiation. Journal of biomedical materials research. Part A, 109(9), 1633-1645.

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Immunofluorescence Staining of MC3T3-E1 Cells

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Fluorescence images of MC3T3-E1 cells on the hydrogels were scoped at 4 days post-seeding. After being fixed by 4% paraformaldehyde for 10 min and permeabilized by 0.2% Triton X-100 for 10 min at room temperature, cells were blocked in 1% bovine serum albumin at 37°C for 30 min. In immunofluorescence, cells were incubated at 37°C with anti-vinculin-FITC antibody (1:50 in PBS, Sigma-Aldrich Co.) for 1 h, rhodamine–phalloidin (RP, 1:200 in PBS, Cytoskeleton Inc) for 1 h, and 4′,6-diamidino-2-phenylindole (DAPI) to visualize cellular focal adhesion, cell cytoskeleton, and cellular nuclei, respectively. The stained MC3T3 cells were immediately viewed using the fluorescent microscope.

Xu H., Liu X., Park S., Terzic A, & Lu L. (2022). Size-dependent osteogenesis of black phosphorus in nanocomposite hydrogel scaffolds. Journal of biomedical materials research. Part A, 110(8), 1488-1498.

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Stem Cell Cytoskeleton Visualization

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The rBMSC stem cells on click-ON scaffolds were fixed, permeabilized, and blocked with 1% bovine serum albumin (BSA). Blocked cells were stained with anti-vinculin−FITC antibody (Sigma-Aldrich Co., Milwaukee, WI) and RP at 37 °C to label vinculin and F-actin, respectively. After staining with DAPI to label nuclei, the cells were imaged on a laser scanning confocal microscope as stated above.

Liu X., Camilleri E.T., Li L., Gaihre B., Rezaei A., Park S., Miller AL I.I., Tilton M., Waletzki B.E., Terzic A., Elder B.D., Yaszemski M.J, & Lu L. (2021). Injectable catalyst-free “click” organic-inorganic nanohybrid (click-ON) cement for minimally invasive in vivo bone repair. Biomaterials, 276, 121014.

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Protein Interaction Detection via Duolink PLA and Immunofluorescence

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Duolink PLA ^®: 5 × 103 OS cells were seeded in Ibidi µ-Slide VI 0.4. 24 later, media was changed to DMEM with 1% FBS. Cells were then fixed with 4% PFA for 15 min at room temperature and incubated overnight at 4 °C with primary antibody against YAP (Cell signaling or Santa Cruz Biotechnology), TEF-1 (Santa Cruz). In situ PLA was performed using DuoLink in Situ Reagents (Sigma-Aldrich) according to the manufacturer’s instructions.
Immunofluorescence assays: cells were seeded onto Ibidi µ-Slide 8 Well overnight, fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton. Samples were incubated with Anti-Vinculin−FITC antibody (Sigma-Aldrich). F-actin and nucleus were stained using respectively Alexa-fluor 488 phalloidin and DAPI. Images were acquired using a confocal microscope (NIKON A1 N-SIM) and processed using ImageJ.

Morice S., Mullard M., Brion R., Dupuy M., Renault S., Tesfaye R., Brounais-Le Royer B., Ory B., Redini F, & Verrecchia F. (2020). The YAP/TEAD Axis as a New Therapeutic Target in Osteosarcoma: Effect of Verteporfin and CA3 on Primary Tumor Growth. Cancers, 12(12), 3847.

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Visualization of Focal Adhesions

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Cells were seeded onto Ibidi µ-Slide 8 Well overnight, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton. The samples were incubated with anti-vinculin−FITC antibody (Sigma-Aldrich, St. Quentin-Fallavier, France) for 2 h at room temperature (1:200) and then washed in phosphate-buffered saline. F-actin and the nucleus were stained using Alexa-fluor 488 phalloidin (1:1,000) and DAPI (1:1,000), respectively. The images were acquired using a confocal microscope (NIKON A1 N-SIM) and processed using ImageJ.

Morice S., Danieau G., Tesfaye R., Mullard M., Brion R., Dupuy M., Ory B., Brounais-Le Royer B., Corre I., Redini F, & Verrecchia F. (2021). Involvement of the TGF-β Signaling Pathway in the Development of YAP-Driven Osteosarcoma Lung Metastasis. Frontiers in Oncology, 11, 765711.

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Analyzing C2C12 Cell Adhesion on CFO/P(VDF-TrFE) Nanocomposites

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For cell adhesion tests, C2C12 cells were seeded at a cell density of 4 000 cells.cm 2 with a drop of 100 µL in 24-well plates and allowed to adhere in the CFO/P(VDF-TrFE) nanocomposites for 45 min at 37 ºC, and then 400 µL of DMEM was added to each well (drop method). After 24 h of culture in BM, C2C12 cells were subjected to immunofluorescence staining in order to analyze the presence of focal adhesions. At this time, the medium of each well was removed and the cells were washed with PBS 1x and fixed in 4% formaldehyde for 10 min at 37 ºC. The nucleus, actin and vinculin were stained by 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), Tetramethylrhodamine B isothiocyanate (TRITC, Sigma-Aldrich) and anti-vinculin-FITC antibody (Sigma-Aldrich), respectively. After this, the samples were again washed with PBS 1x and then incubated with antivinculin-FITC antibody (1:50 in PBS 1x) in the dark at room temperature for 1 h. The cells were subsequently counterstained with TRITC (1:200) and DAPI (1 µg.mL -1 ) at room temperature in the dark for 30 and 5 min, respectively. At the end, the samples were rinsed in PBS 1x and after with distilled water, and finally mounted on slides. The samples were visualized using a fluorescence microscope (Olympus Bx51) with the appropriate filter sets.

Ribeiro S., Ribeiro C., Carvalho E.O., Tubio C.R., Castro N., Pereira N., Correia V., Gomes A.C, & Lanceros-Méndez S. (2020). Magnetically Activated Electroactive Microenvironments for Skeletal Muscle Tissue Regeneration. ACS applied bio materials, 3(7).

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