Antibodies are now routinely used to identify proteins in the central nervous system, to estimate the amounts of different kinds of proteins present, and to determine in which cellular population and subcellular region particular proteins are concentrated. Over the past 8 years, this program project has had a significant component dedicated to the preparation or use of antibodies, but it was limited largely to a single research project. We now propose to expand the use of antibodies in this program to take greater advantage of their potential for use in addressing questions related both to the structure and function of astrocytes and neurons, and in elucidating how these to major cell populations in the brain interact. The purpose of this Core is to provide and characterize immunological reagents, as well as expertise in their use, to all the participants in this program project. Some antibodies, available from commercial sources or from outside collaborators, will be characterized and, if necessary, further purified to allow them to be used to immunoprecipitation, immunoblotting, and immunofluorescence experiments. The Core will also generate its own antibodies. Purified proteins, polypeptide antigens, fusion proteins, and even crude protein mixtures, will be used as antigens for the generation of polyclonal antibodies in rabbits or chickens, and monoclonal antibodies in mice. The Core will select appropriate antigens, preparing then """"""""in house"""""""" whenever possible. Animals will also be injected and boosted """"""""in house"""""""", and pre-immune, and immune sera will be collected. The Core will purify these antibodies as needed, characterize their specificity by ELISA, immunoblotting, and immunoprecipitation, and test their usefulness in immunofluorescence protocols. Experimental assistance in all these techniques, as well as in confocal laser scanning microscopy, will be provided to all participants in the program. We anticipate that all of the projects as well as the Tissue Culture Core in this program project will benefit from the Immunology Core.
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