INTEGRATED ULTRACRYOMICROTOME SYSTEM

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This application requests funds for the purchasing of the components needed to establish an integrated ultracryomicrotome system for the preparation and sectioning of frozen or plastic-embedded biological specimens to be examined by immunoelectron microscopy. This technologically advanced system allows the preservation of fine structural details, while avoiding or minimizing the use of the chemical fixatives, organic solvents or resin polymerization conditions that cause extensive protein denaturation and loss of antigenic epitopes. This make it possible to localize by immunolabeling proteins of interest at the level of resolution attained by electron microscopy. The six investigators that participate in this application are all interested in localizing specific gene products within cultured cells or animal tissues. They work in the areas of protein trafficking (Sabatini/Adesnik, Philips), cell-cell interactions (Cowin, Salzer) and pathogen-host relationships (Zychlinsky, Nussenzweig/Frevert). The system we plan to establish will be located in the electron microscopy suite of the Cell Biology Department, isolated from airdrafts and sudden temperature changes, and will be operated by a skilled and experienced technician, supervised by an accomplished electron microscopist with 15 years experience in immunolabelling techniques. The system we request consists of a Leica UCT ultramicrotome, the Leica FC-S low temperature sectioning attachment, and the Leica equipment necessary for cryopreparation by the rapid freezing technique, and for embedding by cryosubstitution. Funds are also requested to acquire cryotools, an ionizer that minimizes section chattering, and an automatic section stainer.