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Vimentin Antibody in Immunohistochemistry

In a body with thousands of proteins, tissues and enzymes, there is a need to devise a protocol that would allow scientists to locate specific macromolecules to be targeted with other experimental variables. Labeling is one procedure commonly used to attain this goal. Its mechanism is quite simple – it uses substances, such as enzymes that are marked by some radioactive element or fluorescent dye. These labeled enzymes are then allowed to react with specific macromolecules, and the detection can be done using these labels.

Although effective, these enzymes and other labeled molecules can only detect a limited number of targets, thus the need for a better detection molecule. It was Albert H. Coons and his colleagues who first did the labeling of antibodies (IHC Worlds). In 1941, Coons identified pneumococci using an antibody tagged with a fluorescent probe derived from rabbits. A few years later, enzyme labels such as peroxidase and alkaline phosphatase have been introduced (Burnett 83). These various types of labeling gave this technique, known as “Immunohistochemistry”, a great advantage over traditional staining and detection methods.

And since it uses the concept of a specific antibody-antigen reaction, this is more effective and precise than the latter one. It also has a greater number of possible targets; since antigens are commonly found in all tissue and in most macromolecules. For these reasons, immunohistochemistry have been used widely in clinical and experimental laboratories. An experiment or diagnosis using immunohistochemistry depends greatly on the antibody and antigen reaction. Specific locations would require specific antibodies. That is, in order to be effective, one must know the ability of a tissue to express a specific antigen.

Then, a requisite antibody that can detect and react with that antigen is used. Following that, there are a number of antibody groups that would be useful in a specific location or target. One of these antibodies extensively studied and used is the vimentin antibody. Vimentin belongs to the group of intermediate filaments. Intermediate filament proteins are intercellular filaments that form an important part of the cytoskeleton. They measure about 10 mm in diameter, and are divided into six major classes, vimentin being one of these.

Filaments of this type are characteristics of mesenchymal cells, or fibroblasts, and of embryonic, undifferentiated cells. Vimentin antibodies are then useful in determining cell life, lineage, growth progression, and others which may suggest valuable information in studies like tumor growth and metastasis. Vimentin antibodies can be used as any general antibody would be. First, the samples are to be fixed in paraffin. This requires prior fixing to formalin. The samples are then cut thinly using a microtome, and the sheets are placed nicely on slides.

Sometimes, the slides are slightly heated over a flame to allow some of the wax to melt, allowing the sample to adhere to the glass slide. Next, the slides are to be washed. Washing is important, for it removes contaminants and other artifacts that may have formed during the fixation process. This is usually done with xylene, then in ethanol, methanol, isopropanol, then finally in distilled water. Afterwards, the slides are immersed in a blocking solution. Then the samples are incubated with a solution of labeled vimentin antibodies. There are a number of these which can be bought, usually tagged by horseradish peroxidase.

Sometimes, counterstaining is use to further increase the differentiation potential of the sample. By counterstaining the sample, tissues and cells that did not react with the antibody gives off a different color. Viewing can be done using a fluorescence microscope (Brown 261-7). Here is a sample picture of a normal mammary gland stained with vimentin antibodies showing the vimentin positive sites as red (Veerle 31). Figure 1. Vimentin (red) in normal dog mammary gland tissue Immunohistochemistry using vimentin antibodies can be usually seen in studies on the central nervous system.

Diagnostic neuropathology has received numerous benefits from the evolution of this technique. The vimentin antigen is widely expressed in a variety of embryonic and mature tissues found in the central nervous system, thus making its antibody counterpart an appropriate candidate for studies on this field. Most mature neurons do not express this antigen, increasing the specificity of it. By reacting with cells that are “immunoreactive” with it, the antibody helps determine the specific cells expressing the antigen and their location on the body (Ghosh par. 3).

The cells can then be monitored for their growth and localization properties, checking for metastasis and cell lineage. Vimentin is widely used today as a research tool for cancer, such as that regarding malignant melanomas. One problem in studying malignant melanomas is that these can bear a close resemblance to other tumors, cancers and even those of benign ones. They share a number of common histologic features, and other types of staining can cause misdiagnosis and therefore, wrong medication and treatment (Busam par. 1). Differential diagnosis is then required to specifically target these.

Vimentin antibodies are preferred tools in this diagnosis since malignant melanomas usually react with these. This immunoreactivity property of malignant melanomas is unique, and a benign melanoma or a carcinoma would not express this (Zarbo 494). Although some previous studies show that vimentin is still not that specific for immunoreactive reactions with malignant mesotheliomas. Only about 75% of malignant mesotheliomas are found to be immunoreactive with vimentin. Not only that, but also 26% of pulmonary carcinomas are found to show immunoreactivity with vimentin (Jasani 446).

This study suggests that there are still no safe evidences to ensure that vimentin antibodies are specific alone for these malignant tissues, although it gives a sense of the answers these antibodies can bring to the diagnostic field of medicine and pathology. Another promising effect of these antibodies is shown by a study of Falam Veerle. This more recent study used different antibodies in detecting and identifying tumor cells on canines. The researcher found out that although vimentin antibodies are not that 100% specific, they are able to stain and detect tumors strongly compared to other commonly used antibodies (Veerle 50).

In fact, Veerle stated that: “vimentin was the only antibody that stained the tumour cells very clearly positive. Desmin, myosin and NF were generally negative, but in some cases a few single cells were positive. The mouse osteosarcomas that were labelled with SMA, p63 and AE1/AE3, were negative. ” (50-51) These findings show that vimentin antibodies are important tools in pathology and needs to be studied more. Research must be done in order to fine tune the abilities of these antibodies, allowing them to be extremely useful in detecting, localizing, diagnosing, and possibly treating, diseases of the present.

Works Cited

Brown, D. , et al. Histochem Cell Biol 105 (1996): 261-267 Burnett, R. et al. “Immunohistochemistry for light microscopy in safety evaluation of therapeutic agents: an overview”. Toxicology 119 (1997): 83-93. Pub Med website. 28 February 2008 <http://www. ncbi. nlm. nih. gov/pubmed/9129199> Busam, Klaus J. and Barnhill, Raymond L. “Difficult Differentials: Immunologic Stains: When Diagnosis of Melanoma Is Difficult”. Primary Care and Cancer 16 (1996). Cancer Network website. 29 February 2008 <http://www. cancernetwork. com/ journals/primary/p9606g. htm>. Ghosh Mithra. “Immunochemistry in the CNS”. Neurosurgery on the Web.

28 February 2008 <http://www. thamburaj. com/immunohistochemistry. htm>. “Introduction to Immunohistochemistry”. 2007. IHC World; Online Information Center for Immunohistochemistry. 03 March 2008 <http://www. ihcworld. com /intro/intro. htm>. Jasani, Bharat, et al. “The use of vimentin antibodies in the diagnosis of malignant mesothelioma”. Virchows Archiv 406 (1985): 441-448. Veerle, Falam. Immunohistochemical study of canine mammary gland tumors. Upssala University Rudbeck, Sweden: 2005 Zarbo RJ, Gown AM, Visscher DW, et al: “Anomalous cytokeratin expression in malignant melanoma”. Mod Pathol 3 (1990):494-501.

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