Vinculin antibody mouse monoclonal from proteintech validated in western blot wb, immunohistochemistry ihc, immunofluorescence if, flow cytometry fc, enzymelinked. Immunofluorescence protocol paraformaldehyde fixed tissue, see below for nonfixed tissue notes before you begin. Both the vinculin knockdown cells and the y822f rescue cells had reduced levels of ecadherin staining at cellcell junctions fig. Coat coverslips with polyethylineimine or polyllysine for 1 h at room temperature. The intracellular distribution of vinculin and alpha 2. This antibody also reacts with metavinculin, a 145 kda splice variant of vinculin. The role of phosphorylation and limited proteolytic cleavage of talin and vinculin.
Thus, vinculin appears to play a key role in shape control based on its ability to modulate focal adhesion structure and function. Immunofluorescence is a technique that allows the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate fitc. Immunofluorescence is the immunoassay technique that uses a detector antibody or an antigen labeled with florescent dyes lim et al. Ihcicc protocol guide 4 tissue and cell samples must be appropriately harvested and prepared for each ihcicc study. It has also been proposed that vinculin may be involved in the transmembrane induction of actin bundle. Thermo fisher scientific is committed to antibody performance and specificity testing.
Immunofluorescence is a powerful technique that utilizes fluorescentlabeled antibodies to detect specific target antigens. Vinculin antibody nbp241274 analysis of vinculin in a431, daudi, jurkat, raji, and thp1 cell lysate with vinculin antibody at 1 ugml. The specificity of antibodies to their antigen is the base for immunofluorescence. Vinculin monoclonal antibody vinculin antibody sigma. Immunofluorescence protocol university of pittsburgh. Vinculin antibody detects endogenous levels of total vinculin protein. For quantification, we measured the fluorescence intensity at an increasing radius. Sterilize glass coverslips by dipping them in 90% ethanol and carefully drying. Use separate tubs for antibodies and negative control slides. To facilitate the required incubation steps, whole tissues must be cut into ultra thin. Antibody v91 monoclonal anti vinculin by milliporesigma. Protocol for immunofluorescence staining of adhesion cells this is provided as a general protocol. This guide highlights critical steps in the immunofluorescence protocol and demonstrates how protocol changes can.
Vinculin is suggested to be a possible link between ends of the bundles of actin filaments and the plasma membrane. Using a small pair of broadtipped forceps, place the coverslips carefully in the antigen retrieval buffer in the cover glass staining jar, making note. For quantification, we measured the fluorescence intensity at an increasing radius from the bead center, which was normalized to the peak level for each bead so that background levels, instead of peak intensity levels, vary. Vinculin potentiates ecadherin mechanosensing and is. The journal of biological chemistry q 1989 hy the american society for biochemistry and molecular biology, inc. Disregulation of vinculin alters cell adhesion, migration, and growth, which promotes cancer invasion and metastasis.
In mammalian cells, vinculin is a membranecytoskeletal protein in focal adhesion plaques that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton. Aspirate medium, wash cells seeded on clean glass cover slips brie. Cells grown on cover slips or on commercially available incubation chambers. I am new to imagej and i need to quantify immunofluorescence intensity and distribution of protein in the. Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate fitc. Protocol nunc labtek imaging products protocol for growing and staining cells for immunofluorescence enabling cell growth on the nunc labtek ii cc2 chamber slide system other. Good reactivity is obtained with human, bovine, chicken. Lower panel shows costaining of ab155120 green with hoechst 33342 blue.
Recombinant antivinculin antibody epr8185 ab129002. Multicolor iccif can be performed either simultaneously using an antibody cocktail or sequentially by probing one antigen after another. Vinculin is a cytoskeletal protein associated with cellcell and cellmatrix junctions. Download the whole immunofluorescence application guide as a pdf here.
To demonstrate the variability in staining patterns, we exam ined the effect of different fixation and extraction procedures on the localization of vinculin and alpha, q integrin on cul tured cells and whole tissue. In another example, the optimal protocol for staining a low abundance protein in a methanol fixed, frozen. This can be performed either simultaneously in a mixture or sequentially one antigen after another. Place slides in a glass slide holder and fill in the rest of the rack with blank. Immunofluorescence protocol adherent and suspension for. Immunofluorescence uses a fluorescent label, usually fluorescein, conjugated either to the antiviral antibody itself direct immunofluorescence or to an antiantibody indirect immunofluorescence, and viewed with ultraviolet light fig.
Simultaneous and sequential staining follow the same basic protocol, but considerable variation exists in the blocking and antibody incubation steps. It functions as one of several interacting proteins involved in anchoring factin to the membrane. A cytoskeletal protein associated with cellcell and cellmatrix interactions. By immunofluorescence if, we noticed that vinculin and factin are already recruited to unforced beads. Vinculin phosphorylation differentially regulates mechanotransduction at cellcell and cellmatrix adhesions.
Immunofluorescence an overview sciencedirect topics. For optimum staining, incubations should be carried out on a slowmoving rotary shaker unless the cell line being used is delicate. Nptx2 promotes colorectal cancer growth and liver metastasis by the activation of the canonical wntbetacatenin pathway via fzd6. Seed adherent cells on 6well tissue culture plates in a sterile tissue culture hood. Western blot analysis of extracts from various cell lines using vinculin e1e9v xp rabbit mab upper or. Immunofluorescence is a valuable auxiliary diagnostic tool for autoimmune bullous diseases. Reliable double immunofluorescence labeling for confocal laser scanning microscopy requires good separation of the signals generated by the fluorochromes. Tissue sample homologous to the test sample incubated with an antibody isotype not specific for vinculin. This antibody reacts with human, mouse, rat, pig samples. Immunofluorescence immunofluorescence is an antigenantibody reaction where the antibodies are tagged labelled with a fluorescent dye and the antigenantibody complex is visualized using ultra. Immunofluorescence microscopy pdf immunofluorescence microscopy is used to localize specific constituents in tissue sections or immobilized cells using fluorescent tags as labels. Optimization of concentration or incubation condition of the primary antibody and the secondary.
Cell lines grow cultured cells on sterile glass cover slips or slides overnight at 37. Jun 28, 2010 by immunofluorescence if, we noticed that vinculin and factin are already recruited to unforced beads. Please refer to proscis tissue immunofluorescence staining protocol for materials, and easy steps for deparaffinization rehydration, antigen retreval, blocking, primary antibody preparation, biotinylated secondary antibody preparation and detection. Vinculin antibody hvin 1 nb6001293 double labeling of cultured chicken fibroblasts for vinculin using monoclonal antihuman vinculin and rabbit anti. Immunofluorescence immunofluorescence is an antigenantibody reaction where the antibodies are tagged labelled with a fluorescent dye and the antigenantibody complex is visualized using ultraviolet fluorescent microscope. Blocking solution 1% bsa or 10% fcs fetal calf serum in.
The amino acid sequence of human vinculin has been determined. Immunofluorescence protocol all steps in this protocol are performed at room temperature unless otherwise indicated. This can be done by heating the buffer in a coverglass staining jar which is placed in a water bath at 95c. Vinculin regulates cellsurface ecadherin expression by. The antibodyantigen complex is labeled with any of a variety of fluorochromes emitting light from the near uv to the near ir. Icc and if protocol multicolor immunostaining optional step to examine the codistribution of two or more different antigens in the same sample, use a double immunofluorescence procedure.
Immunohistochemical analysis of paraffinembedded human colon carcinoma using vinculin e1e9v xp rabbit mab performed on the leica bond rx. Fluorochromes are dyes that absorb ultraviolet rays and emit visible light. In our hands, for best results, samples should be paraformaldehyde fixed and frozen using 2methylbutane cooled with liquid nitrogen see tissue fixation and freezing protocol. The protein consists of 1066 amino acid residues and. Immunocytochemistry and immunofluorescence protocol. Davidson the ohio state university, biology department. Immunofluorescence staining protocol from ihc world 1. Validated in wb, ip, flow cyt, iccif and tested in mouse, rat, human. Get started quickly and move forward confidently using your new if resource guide. To support this commitment, each invitrogen antibody that is indicated for immunofluorescence applications has been tested using a protocol. Immunocytochemistry and immunofluorescence protocol abcam.
Immunocytochemistry immunofluorescence anti vinculin antibody ab155120 immunofluorescent analysis of paraffin methanolfixed mcf7 cells, labeling vinculin with ab155120 at 1500 dilution. Cells were seeded onto coverslips for 24 h and incubated for 40 min with beads. Our results suggest that vinculin may play specific roles in the emt and metastasis of crc and that loss of vinculin. The focal adhesion protein antibody sampler kit provides an economical means to evaluate proteins. Vinculin antibody nbp241274 staining of vinculin in jurkat cells with vinculin. Fixative 4% formaldehyde in pbs freshly prepared 3. Vinculin antibody mouse monoclonal from proteintech validated in western blot wb, immunohistochemistry ihc, immunofluorescence if, flow cytometry fc, enzymelinked immunosorbent assay elisa applications. Immunofluorescence techniques journal of investigative. Vinculin was identified as a component of adherens junctions 30 years ago, yet its function there remains elusive. Vinculin binds to talin, paxillin, and alphaactinin.
Monoclonal anti vinculin specifically stains vinculin at cellcell and cellsubstrate contacts in tissue and cultured cells using indirect immunofluorescent labeling. This guide highlights critical steps in the immunofluorescence protocol and demonstrates how protocol. Protocol for immunofluorescence staining of adhesion cells. Applications key ab array antibody array ae affinity electrophoresis affin chroma. Immunofluorescence is a powerful tool for elucidating the complex signaling events that underlie biological processes and disease. Please refer to the applications section on the front page of product datasheet or product webpage to determine if this product is validated and approved for use on cultured cell lines ific, paraffinembedded samples ifp, or frozen tissue sections iff. Vinculin was identified in tissue sections by immunofluorescence using an anti vinculin. The researcher should optimize the precise conditions for a particular assay. Vinculin vcl is a cytoskeletal protein associated with cellcell and cellextracellular matrix adherenstype junctions. The antibody reacts with the 116 kda vinculin band in immunoblotting.
A transmembrane relationship between fibronectin and. Vinculin s ability to interact with integrins to the cytoskeleton at the focal adhesion appears to be critical for control of cytoskeletal mechanics, cell spreading, and lamellipodia formation. Immunofluorescence is an assay which is used primarily on biological samples and is classically defined as a procedure to detect antigens in cellular contexts using antibodies. For western blots, incubate membrane with diluted primary antibody in 5% wv bsa, 1x tbs, 0. Protocols and troubleshooting tips frequently asked questions we have a policy of honesty and so we share everything that we know about our. However, this approach removes vinculin from both cellmatrix and cellcell adhesions, making it impossible to distinguish its contribution at each site. Oct, 2019 a cytoskeletal protein associated with cellcell and cellmatrix interactions. Vinculin antibody hvin 1 nb6001293 primary human osteoblast cells labeled red, actin was stained with phalloidin green and nuclei stained blue. It is used widely in both scientific research and clinical laboratories.
At vector laboratories, simplicity, consistency, reproducibility, and sensitivity are all inherent qualities of our reagents and workflows. Immunofluorescence protocol united states biological. Vinculin monoclonal antibody vinculin antibody sigmaaldrich. Immunocytochemistry and immunofluorescence protocol procedure for staining of cell cultures using immunofluorescence. Optimization of concentration or incubation condition of the primary antibody and the secondary antibody for your own specimen is necessary. Antibody v91 monoclonal antivinculin by milliporesigma. Immunofluorescence is usually performed on frozen tissue sections fixed in acetone or methanol. Deletion studies are consistent with the idea that vinculin is important for the organization of cellcell junctions. Apr 28, 2014 to determine if the morphological changes were due to an effect on ecadherin, immunofluorescence was used to examine the localization of ecadherin in confluent cell monolayers. Immunofluorescence microscopy protocol immunofluorescence microscopy is used to localize specific constituents in tissue sections or immobilized cells using fluorescent tags as labels.
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