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. Immunofluorescence protocol for tissue sections note. Confocal immunofluorescence analysis of methanolfixed hela human epithelial cell line from cervix adenocarcinoma cells labeling vimentin, using ab7321 green at a 1500 dilution. Monoclonal anti vimentin antibody can be used in immunofluorescent labeling of frozen tissue sections to localize vimentin in fibroblasts, endothelial cells, lymphoid tissues and melanocytes. Pdf relationship of antivimentin antibodies to anti. Immunofluorescent staining of cells antibody resource. This section of the guide will highlight some of the data we use to support our if protocol recommendations. A direct immunofluorescence the best site and evolution time of skin lesions to perform biopsy for direct immunofluorescence examination dif depend on the disease under investigation.
This guide highlights critical steps in the immunofluorescence protocol and. For vimentin stain, i add in a permeabilization step here with 0. Immunofluorescence study of intermediate filament protein. Basic iccif protocol a standard iccif protocol involves fixation, permeabilization, blocking, immunolabeling, counterstaining, and microscopic imaging of stained cells see the flow chart in figure 2. Provided below are standard protocols that you may find useful for product applications.
Each step of the iccif protocol requires optimization as experimental variables in each step can significantly impact staining outcome. If proteins are found in all animal cells as well as bacteria. Immunofluorescence stained samples are examined under a fluorescence microscope or confocal microscope. Primary antibodies secondary antibodies staining dyes kits. We have tested common variations in fixation, permeabilization, and antibody concentration for many of our ifapproved antibodies. Vimentin staining green, factin staining with phalloidin red and nuclei with dapi blue is shown. Because fluorescent dyes such as fluorescein and rhodamine can be coupled to. Generally, the biopsy should have an appropriate extension 4 mm punch and depth that involves both the epidermis and dermis in sufficient proportion. Immunofluorescence protocol for frozen tissue download a pdf. Protocol for emulate organchips immunofluorescence staining. Only for research applications, not for diagnostic or therapeutic use. Because fluorescent dyes such as fluorescein and rhodamine can be coupled to antibodies without destroying their specificity, the conjugates can complex with antigen and be visualized via fluorescence microscopy. Exclusively from the antibody resource, you can buy this superstarter kit, which is a collection of trial sizes of these superstar vimentin antibodies highly rated and independently tested by us, to test using the specific experimental conditions which your research dictates, so that you can identify which will be the most appropriate for your research project.
The current protocol the custom version will disappear and be replaced with the readytouse rtu version if the custom protocol is not renamed. Recommended fixative and incubation time 4% pfa, 15 mins at room temperature recommended permeabilization and incubation. This monoclonal antibody reacts with the intermediate filament protein vimentin present in cells of mesenchymal origin. Vimentin label for immunostaining of vimentin intermediate filaments for immunofluorescence of vimentin intermediate filaments in fixed cells. Immunofluorescence microscopy pdf immunofluorescence microscopy is used to localize specific constituents in tissue sections or immobilized cells using fluorescent tags as labels. A blocking step was performed using 1% bsapbs for 1 hour. The antibodyantigen complex is labeled with any of a variety of fluorochromes emitting light from the near uv to the near ir. Vimentin antibodies for immunofluorescence antibody. The optimum antibody dilution and protocols for a specific application can vary. The fibroblastic cells contain only vimentin and so are. Vimentin is an intermediate filament that is ubiquitously expressed in mesenchymal cells such as fibroblasts, smooth muscle cells. Vimentin is useful as part of an antibody panel for differential diagnostic purposes, such as differentiating soft tissue tumors.
Basic iccif protocol a standard iccif protocol involves fixation, permeabilization, blocking, immunolabeling, counterstaining, and microscopic imaging of stained cells see the flow chart in. Immunocytochemistry and immunofluorescence protocol abcam. This technique uses the specificity of antibodies to their. Vimentinlabel for immunostaining of vimentin intermediate. Vimentin antibody used for immunofluorescence on cryosections of mouse, canine and human quadriceps muscle at a dilution of 1.
Vimentin is a type iii intermediate filament if protein that is expressed in mesenchymal cells. For tissue stored at 80c, remove from freezer and equilibrate at 20c for about 15 minutes before sectioning. In our hands, for best results, samples should be paraformaldehyde fixed and frozen. Vimentin antibody additional information gene id 7431 other names vimentin, vim targetspecificity this vimentin antibody is generated from rabbits immunized with human vimentin recombinant. Exclusively from the antibody resource, you can buy this superstarter kit, which is a collection of trial sizes of these superstar vimentin antibodies highly rated and independently tested by us, to test. Cells grown on cover slips or on commercially available incubation chambers. Humanmouserat vimentin apcconjugated antibody ic2105a. Protocol for emulate organchips immunofluorescence. The new protocol names are listed in the table below. This application protocol describes a method for the immunofluorescence staining of cells enriched from a human blood sample with vortex device. Immunofluorescence microscopy pdf immunofluorescence microscopy is used to localize specific constituents in tissue sections or. Vimentin antibody additional information gene id 7431 other names vimentin, vim targetspecificity this vimentin antibody is generated from rabbits immunized with human vimentin recombinant protein. Western blot analysis of extracts from control hela cells lane 1 or vimentin knockout hela cells lane 2 using vimentin 5g3f10 mouse mab upper or.
This guide highlights critical steps in the immunofluorescence protocol and demonstrates how protocol changes can affect the final outcome of your experiment. The current protocol the custom version will disappear and be. Ctcs were categorized as epithelial epictc if they were vimentinnegative and. An optimized protocol is necessary to achieve consistent, reliable if results. Rapid multiple immunofluorescent staining for the simultaneous detection of cytokeratin and vimentin in the cytology of canine tumors article in veterinary clinical pathology 472 march 2018. Immunofluorescence protocol united states biological. Monoclonal antivimentin antibody has been used in western blotting and immunocytochemistry. Introduction the vimentin filament network plays a key role in cell architecture and signaling in mesenchymal cells. This cell population was disposed along a gradient. Visualize vimentin via fluorescent imaging in fixed emulate organchips. The clinical interpretation of any staining or its.
Only for research applications, not for diagnostic or. Western blot analysis of vimentin using a 20 g 293t whole cell lysate b 10 g 293t whole cell lysate c 5 g 293t whole cell lysate and d 1 g 293t whole cell lysate. Antivimentin antibodies were detected by indirect immunofluorescence in hep2 cells, and levels of antivimentin antibodies were measured using an enzymelinked immunosorbent assay. Vimentin is the main intermediate filament protein in mesenchymal. View specific protocols for vimentin antibody nb300223. 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.
Immunofluorescence microscopy protocol immunofluorescence microscopy is used to localize specific constituents in tissue sections or immobilized cells using fluorescent tags as labels. Immunofluorescence protocol university of pittsburgh. Please optimize the procedure since experimental conditions can vary for different samples. 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. Get your team aligned with all the tools you need on one secure, reliable video platform.
Immunofluorescence is an antigenantibody reaction where the antibodies are tagged labelled with a fluorescent dye and the antigenantibody complex is visualized using ultraviolet fluorescent. Component of the vimentin superstarter antibody panel. Immunocytochemistry and immunofluorescence protocol. The absence of signal in the vimentin knockout hela cells confirms specificity of the antibody for vimentin. Increase the manual exposure time of your camera or use the autoexposure option that will adjust. Use separate tubs for antibodies and negative control slides. Immunocytochemistry icc handbook novus biologicals.
A172 human glioblastoma cell line was stained with rat antihuman mouserat vimentin apcconjugated monoclonal antibody catalog. Blocking solution 1% bsa or 10% fcs fetal calf serum in. Protocol immunofluorescent analysis of hela cells human epithelial cells from cervix adenocarcinoma and skov3 cells human epithelial cells from ovarian carcinoma was performed using a leica tcs sp2 confocal laser scanning microscope. Immunofluorescence if combines the use of antibodies with fluorescence imaging.
Monoclonal antivimentin antibody produced in mouse clone v9. Vimentin is useful as part of an antibody panel for differential diagnostic purposes, such as differentiating soft tissue. This protocol is using an antibody cocktail against human. Vimentin is an important cytoskeleton protein for the biological function of macrophages. Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. If, along with tubulinbased microtubules and actinbased microfilaments, comprises the cytoskeleton. Antivimentin antibody v9 cytoskeleton marker ko tested. Visit the supplier page for more detailed antibody product information, such as specificity and validated applications. Place slides in a glass slide holder and fill in the rest of the rack with blank. Protocol recommendations intellipath and manual use peroxide block. Immunofluorescence protocol paraformaldehyde fixed tissue, see below for nonfixed tissue notes before you begin. For immunofluorescence of vimentin intermediate filaments in fixed cells. Broad interspecies crossreactivity has been observed with human, rat, and chicken vimentin. Vimentin is an intermediate filament that is ubiquitously expressed in mesenchymal cells such as fibroblasts, smooth muscle cells, and endothelium.
Immunofluorescence detection of the vimentin epitope in chromatin. 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. Vimentinlabel for immunostaining of vimentin intermediate filaments for immunofluorescence of vimentin intermediate filaments in fixed cells. This protocol is using an antibody cocktail against human vimentin, ncadherin and cytokeratin. If, along with tubulinbased microtubules and actin. Vimentin was detected in immersion fixed k562 human chronic myelogenous leukemia cell line but is not detected in vimentin. Fixative 4% formaldehyde in pbs freshly prepared 3.
Examples of staining using traditional icc, iccif, and ihc with if based detection. Mouse anti vimentin antibody reacts specifically with vimentin. This can be performed either simultaneously in a mixture or sequentially one antigen after another. Monoclonal antivimentin antibody can be used in immunofluorescent labeling of frozen tissue sections to localize vimentin in fibroblasts, endothelial cells, lymphoid tissues and melanocytes. Western blot blocking peptides dot blot immunohistochemistry immunofluorescence immunoprecipitation flow cytomety cell culture retrieval was by heat mediation with a edta buffer ph9.
Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody. Vimentin monoclonal antibody western, immunofluorescence. Tested in western blot wb, immunofluorescence if, immunocytochemistry icc and immunohistochemistry paraffin ihc p. Vimentin specificity is shown by immunocytochemistry in knockout cell line. Vimentin label is a small vimentin binding protein derived from a camelid vhh socalled nanobody and conjugated to the fluorescent dye atto 488 from attotec. In this application guide, we will highlight the critical steps in our protocol for if, introduce important concepts about antibody performance and design of controls, and provide supporting data to explain our recommendations.
Immunofluorescence study of intermediate filament protein vimentin of cpactivated macrophages. Vimentin antibody staining protocol for immunohistochemistry. This cell population was disposed along a gradient figure 3 characterized by low density within cortex 35 cellsmm 2 to higher densities in osom 100 cellsmm. Alphatubulin filaments were labelled with an antialpha tubilin antibody red at a 12000 dilution. Detection of vimentin in a172 human cell line by flow cytometry. Immunostaining of cells in tissue culture protocol specific for vimentin antibody nb300223 find general support by application which include. Follow the instructions to resolve the protocol name conflict. Introduction immunofluorescence techniques referenced in this guide. Monoclonal antivimentin antibody produced in mouse has been used as a primary antibody to localize vimentin by immunohistochemistry ihc and for immunoprecipitation of vimentin. Multicolor iccif can be performed either simultaneously using an antibody cocktail or sequentially by probing one antigen after another. Development of a multiparameter immunofluorescence. The vimentin antigen is a documented cancer and neural stem cell marker. Protocol immunofluorescence microscopy is used to localize specific constituents in tissue sections or immobilized cells using fluorescent tags as labels.
Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Immunohistochemistry on formalin fixed paraffin embedded human tissue. Protocol immunofluorescent analysis of hela cells human epithelial cells from cervix adenocarcinoma and. K confocal if analysis of vimentinpositive snb19 using vimentin d21h3. Nucleated cells that were epithelial markerpositive and exclusion markernegative were identified as ctcs. Simultaneous and sequential staining follow the same basic protocol, but considerable variation exists in the blocking and antibody incubation steps. In controls, vimentinpositive fusiform interstitial cells were present in the different zones of renal interstitium figures figures3 and 3 and 5a.
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