Primary anti vinculin antibody

Manufactured by Merck Group

Sourced in United States

The Primary anti-vinculin antibody is a laboratory reagent used for the detection and quantification of vinculin, a protein that plays a crucial role in cell-cell and cell-matrix adhesion. This antibody can be used in various immunological techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and localization of vinculin in biological samples.

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Vinculin (439 citations) Anti-vinculin (287 citations) Anti-vinculin antibody (51 citations) Vinculin antibody (27 citations) Primary mouse anti-vinculin antibody (9 citations) Vinculin primary antibody (2 citations) Mouse monoclonal anti-vinculin primary antibody (2 citations)

8 protocols using primary anti vinculin antibody

Visualizing Focal Adhesions and Stress Fibers

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Ovarian and breast cells were grown on gradient structures between 16 to 24 hours prior to staining. These cells were fixed in with 4% paraformaldahyde in PBS for 10 minutes. Following a three-step wash with 1X PBS, the cells were permeabilized with 0.2% Triton X-100 and treated with a 5% BSA blocking solution for 45 minutes. To stain for focal adhesions, the cells were incubated with an anti-vinculin primary antibody (at 1:200 dilution; EMD Millipore) overnight at 4°C followed by incubation with a fluorescent secondary antibody, IgG Alexa488 (1:1000 dilution; Invitrogen), for 1 hour at room temperature. In addition, the cells were stained for f-actin to visualize stress fibers using Texas Red conjugated phalloidin (Invitrogen).
Cells were treated with a DAPI/DABCO anti-fade solution to minimize photobleaching during fluorescent imaging. Fluorescent images of each respective channels were collected with a highly sensitive CCD camera (QImaging R2000R) using a 40X 0.8NA water immersion lens.

Ajeti V., Lara-Santiago J., Alkmin S, & Campagnola P.J. (2017). Ovarian and Breast Cancer Migration Dynamics on Laminin and Fibronectin Bi-directional Gradient Fibers Fabricated via Multiphoton Excited Photochemistry. Cellular and Molecular Bioengineering, 10(4), 295-311.

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Immunofluorescent Analysis of Cytoskeleton and Focal Adhesions

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Cells were plated in confocal dishes for two days followed by fixation in 4% paraformaldehyde at room temperature for 20 min. After permeabilization with 0.1% Triton X-100 for 5 min, cells were incubated in 1%BSA in PBS for 1 h at room temperature. Cells were then incubated overnight with primary antibodies against ZO-1 (1:500, Proteintech) followed by incubation with goat anti-Rabbit Secondary Antibody, Alexa Fluor 488 (1: 5000, Invitrogen), and TRITC-conjugated Phalloidin (1:1000, Sigma-Aldrich). Staining of focal adhesion was conducted with the Actin Cytoskeleton and Focal Adhesion Staining Kit (Merck Millipore). For antigen staining, cells were incubated with anti-vinculin primary antibody (1:200, Merck Millipore) overnight at 4 °C, followed by incubation with a secondary FITC-conjugated antibody (1:5000, Proteintech) and TRITC-conjugated Phalloidin (1:1000, Sigma-Aldrich) for 1 h, respectively. Nuclei counterstaining was performed by incubating cells with DAPI for 5 min at room temperature. The quantification of focal adhesion (FA) number, actin and ZO-1 intensity were determined using ImageJ software (Version 1.8.0). Original data were displayed in supplementary data.

Ji C., Zhang M., Hu J., Cao C., Gu Q., Liu Y., Li X., Xu D., Ying L., Yang Y., Gao H., Li J, & Yu L. (2022). The kinase activity of integrin-linked kinase regulates cellular senescence in gastric cancer. Cell Death & Disease, 13(7), 577.

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Quantifying Cell-Scaffold Interactions using Fluorescence Microscopy

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Ovarian cells were grown on fabricated structures between 16 to 24 hours prior to staining. These cells were fixed in with 4% paraformaldahyde in PBS for 15 minutes. Following two washes with 1x PBS, the cells were permeabilized with 0.3% Triton X-100 for 10 minutes and stained with Texas Red conjugated phalloidin for 30 minutes. At least 8–12 cells were analyzed for each cell/scaffold combination. CurveAlign was used to quantify the angular distribution of f-Actin fibers for cells in a given pattern as well as the overall collagen alignment from the SHG images [62 (link)]. To stain for focal adhesions, the cells were incubated with an anti-vinculin primary antibody (at 1:200 dilution; EMD Millipore) overnight at 4°C followed by incubation with a fluorescent secondary antibody, IgG Alexa488 (1:1000 dilution; Invitrogen) for 1 hour at room temperature. The number of focal adhesions per cell and integrated areas were determined in ImageJ.

Alkmin S., Brodziski R., Simon H., Hinton D., Goldsmith R.H., Patankar M, & Campagnola P.J. (2019). Migration dynamics of ovarian epithelial cells on micro-fabricated image-based models of normal and malignant stroma. Acta biomaterialia, 100, 92-104.

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Isolation and Characterization of Monocyte-Derived Macrophages

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Frozen peripheral blood mononuclear cells (PBMCs) from healthy donors and patients were thawed and CD14⁺ cells selected using magnetic beads (Miltenyi). 2 x 10⁵ cells/ well in a 24 well plate were seeded on 10ug/ml fibronectin-coated cover slips (R&D systems) in 500ul 20ng/ml macrophage colony stimulating factor (MCSF, Gibco) for 6 days to obtain monocyte-derived macrophages (MDMs). Cells were fixed with paraformaldehyde 4% (Thermo Fisher Scientific) for 10 minutes on ice followed by 8% for 20 minutes at room temperature, permeabilised with 0.1% triton (Sigma) for 5 minutes at room temperature and non-specific binding reduced by blocking with 5% BSA/PBS for 1 hour at room temperature. Cells were incubated with primary anti-vinculin antibody (Sigma 1:200) for 1 hour at room temperature, washed twice with PBS and incubated with secondary antibody conjugated to Alexa Fluor 488 (1:500 Life Technologies) and phalloidin-conjugated to Alexa Fluor 633 (1:200 Thermo Fisher Scientific) for one hour at room temperature. Cells were washed twice with PBS and cover slips mounted onto slides using mounting solution with DAPI for nuclear staining (ProLong Diamond Antifade Mountant with DAPI, Life Technologies) overnight. Slides were imaged using Zeiss 710 confocal microscope at 63x magnification and podosome analysis was carried out on at least 100 cells per sample from 10 fields of view.

Thaventhiran J.E., Allen H.L., Burren O.S., Rae W., Greene D., Staples E., Zhang Z., Farmery J.H., Simeoni I., Rivers E., Maimaris J., Penkett C.J., Stephens J., Deevi S.V., Sanchis-Juan A., Gleadall N.S., Thomas M.J., Sargur R.B., Gordins P., Baxendale H.E., Brown M., Tuijnenburg P., Worth A., Hanson S., Linger R., Buckland M.S., Rayner-Matthews P.J., Gilmour K.C., Samarghitean C., Seneviratne S.L., Sansom D.M., Lynch A.G., Megy K., Ellinghaus E., Ellinghaus D., Jorgensen S.F., Karlsen T.H., Stirrups K.E., Cutler A.J., Kumararatne D.S., Chandra A., Edgar J.D., Herwadkar A., Cooper N., Grigoriadou S., Huissoon A., Goddard S., Jolles S., Schuetz C., Boschann F., Lyons P.A., Hurles M.E., Savic S., Burns S.O., Kuijpers T.W., Turro E., Ouwehand W.H., Thrasher A.J, & Smith K.G. (2020). Whole genome sequencing of a sporadic primary immunodeficiency cohort. Nature, 583(7814), 90-95.

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Isolation and Characterization of Monocyte-Derived Macrophages

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Visualizing Cell Adhesion on Titanium

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The PC12 cells were incubated with the Ti substrate for 1 day. After this time, the samples were initially fixed with 4% paraformaldehyde for 15 min, permeabilised in 0.1% Triton X for 5 min then blocked with 1% Bovine Serum Albumin (BSA) for 60 min. Image-IT^® FX Signal Enhancer (Invitrogen, Carlsbad, CA, USA) was also used during the fixation stage to enhance the subsequent fluorescent signals. Samples were then treated with a primary anti-vinculin antibody (Sigma, St. Louis, MO, USA) overnight, followed by goat anti-mouse secondary antibody conjugated with Alexa Fluor 594 (Invitrogen). Actin filaments were visualised by staining the cells with Alexa Fluor 488 conjugated phalloidin (Invitrogen). Cell nuclei were labelled using TO-PRO3 (Invitrogen). To study the extent of cell differentiation after 5 and 7 days of incubation, the anti-nestin antibody (Sigma) was applied as the primary antibody. Samples were then imaged using a Fluoview FV10i microscope (Olympus, Tokyo, Japan) at 60× magnification.

Wandiyanto J.V., Linklater D., Tharushi Perera P.G., Orlowska A., Truong V.K., Thissen H., Ghanaati S., Baulin V., Crawford R.J., Juodkazis S, & Ivanova E.P. (2018). Pheochromocytoma (PC12) Cell Response on Mechanobactericidal Titanium Surfaces. Materials, 11(4), 605.

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Cytoskeletal Protein Staining of HeLa Cells

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For the cytoskeletal protein staining, HeLa cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and incubated with the primary anti-vinculin antibody (Sigma), an anti-mouse IgG CY3 conjugate developed in sheep (Sigma), an anti-NMMIIA (Abcam) and the Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen). All of the incubations were performed for 30 min at 37°C. To stain actin SFs, cells were incubated with Oregon Green 448 phalloidin (Invitrogen) for 15 min at 37°C. The stained cells for each preparation were mounted with Vectashield Mounting Medium with DAPI (Vector Laboratories). The samples were analyzed using the 63× objective of an Axiovert 200 M confocal LSM 510 META Zeiss microscope.

Elosegui-Artola A., Jorge-Peñas A., Moreno-Arotzena O., Oregi A., Lasa M., García-Aznar J.M., De Juan-Pardo E.M, & Aldabe R. (2014). Image Analysis for the Quantitative Comparison of Stress Fibers and Focal Adhesions. PLoS ONE, 9(9), e107393.

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TMP-Induced Actin and Vinculin Dynamics

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Serum-starved cells were cultured for 24 h in the presence of 100,000 TMPs obtained from untreated or PTX-treated cells, as indicated in the text. In some experiments, TMPs were cultured with cells for 24 h in serum-free medium in the presence of anti-CD44 antibodies (1 μg/mL). Cells were rigorously washed in PBS (100-fold volume), and then seeded on fibronectin-coated plates (20 µg/mL fibronectin, Biological Industries). After 4 h, cells were fixed using 4% paraformaldehyde (PFA) and immunostained with a primary anti-vinculin antibody (1:100, Sigma-Aldrich) and Cy2-conjugated secondary antibody. Actin was stained with Alexa 488 conjugated phalloidin (1:100, Invitrogen, Carlsbad, CA, USA). Images were acquired with a LSM 700 Zeiss confocal microscope (Zeiss Ltd. Oberkochen, Germany).

Shechter D., Harel M., Mukherjee A., Sagredo L.M., Loven D., Prinz E., Avraham S., Orian-Rousseau V., Geiger T., Shaked Y, & Wolfenson H. (2020). Breast Cancer-Derived Microparticles Reduce Cancer Cell Adhesion, an Effect Augmented by Chemotherapy. Cells, 9(10), 2269.

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