My task as an intern would be to complete a small project within the context of Surya’s and Gill’s research. Each of the postdocs takes a slightly different approach to detecting the subtle differences between mutant and wild-type nerve cells. Surya uses immunocytochemistry (IC), a method of staining cells with antibodies so that she can pinpoint the location of the huntingtin protein, for example, in the nuclei. Meanwhile, Gill uses immunohistochemistry (IH), a method of staining tissue slices (from the striatum, in this case) with the same antibodies, also to locate huntingtin in the nerve cells.
Antibodies are proteins made by the body’s immune system as a defense against foreign material, such as bacteria or viruses, which enters the body. These Y-shaped proteins attack and neutralize the substances, called antigens, that triggered the immune response. Each antibody has a specific antigen to which it binds. The IC and IH methods make use of an antibody’s ability to recognize a particular antigen, rather than its ability to attack and neutralize it. Please see below for a diagram of an antibody.
Fig. 4. The structure of an antibody. (from Wikipedia.)
To visualize the location of the huntingtin protein in the nucleus of a mouse nerve cell, researchers use a technique called immunostaining as part of the IC and IH methods. After fixating, or preserving, a cell sample or tissue slice on a cover slip or slide, they add a small amount of a primary antibody. The primary antibody recognizes and binds to a specific place on the huntingtin protein’s surface, called an epitope. Then, a secondary antibody that comes from another animal is used to detect the first. The secondary antibody contains a fluorescent molecule, which allows the researchers to see the position of the huntingtin in the cell under the powerful confocal microscope. Multiple secondary antibodies bind to the primary, thereby amplifying the fluorescent signal. Please see below for a schematic diagram of immunostaining.
Fig. 5. Immunostaining.
The primary antibody recognizes the polyglutamine tract of the huntingtin protein, and the secondary antibody recognizes the primary. The secondary antibody contains a fluorescent molecule to help scientists visualize the huntingtin under a microscope. Multiple secondary antibodies bind to the primary for fluorescent signal amplification.
Using confocal microscopy, scientists can visualize the huntingtin protein molecules to which the primary antibody binds. They can therefore show the localization of huntingtin and make conclusions as to the site-specific function of the protein (both mutant and wild-type) in the cells.
My project fit neatly into this experimental framework and had two objectives. The wet lab component of the project, performed at the lab bench, involved immunostaining with a primary antibody made at two different commercial laboratories. My goal was to determine which of the two versions was better, and under what conditions, for seeing differences between the wild-type and mutant cells with regard to the staining pattern. See below for a picture of me performing an immunostaining procedure.
Fig. 6. Taylor Altman performs an immunostaining procedure.
She applies a primary antibody stain to mutant and wild-type cells on cover slips.
The dry lab component, performed at my desk, entailed building an electronic database of information about huntingtin antibodies. In a Microsoft Excel spreadsheet, I organized the information by such categories as antibody name, epitope, and animal host (the animal from which the antibody is taken). The database will eventually be turned into a website for use by HD researchers all over the world.
Last Modified: 05/22/2009
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