INTEGRATED ULTRACRYOMICROTOME SYSTEM

这项申请要求提供资金，用于购买建立综合超低温显微系统所需的部件，以制备和切片将由免疫电子显微镜检查的冷冻或塑料包埋的生物标本。这一技术先进的系统可以保存精细的结构细节，同时避免或最大限度地减少使用化学固定剂、有机溶剂或树脂聚合条件，这些条件会导致蛋白质的广泛变性和抗原表位的丧失。这使得通过免疫标记感兴趣的蛋白质在电子显微镜获得的分辨率水平上进行定位成为可能。参与这项申请的六名研究人员都对在培养细胞或动物组织中定位特定基因产物感兴趣。他们在蛋白质贩运(Sabatini/Adesnik，飞利浦)、细胞-细胞相互作用(Cowin，Salzer)和病原体-宿主关系(Zychlinsky，Nussenzweig/Frevert)领域工作。我们计划建立的系统将位于细胞生物系的电子显微镜套间，与空气和突然的温度变化隔离，并将由一名熟练和经验丰富的技术人员操作，由一名在免疫标记技术方面拥有15年经验的熟练电子显微镜专家监督。我们所要求的系统包括徕卡UCT超微切片机，徕卡FC-S低温切片附件，以及通过快速冷冻技术进行冷冻修复和通过冷冻置换进行包埋所需的徕卡设备。还需要资金来购置冷冻工具、可最大限度地减少切片抖动的电离器和自动切片着色器。

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.