Cryosectionning unit for an ultramicrotome to facilitate immunolabeling for electron microscopy and protein localization using mass spectrometry

用于超薄切片机的冷冻切片装置，可促进电子显微镜免疫标记和使用质谱法进行蛋白质定位

• 批准号: 390535-2010
• 负责人: Page, Louise
• 金额: $ 3.89万
• 依托单位: University of Victoria
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2009
• 资助国家: 加拿大
• 起止时间: 2009-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=443213
• 关键词: Cryosectionning; unit; ultramicrotome; facilitate; immunolabeling

Five co-applicants on this proposal would like to purchase an attachment for our existing ultramicrotome, which was funded by NSERC in 2003, to allow us to cut very thin slices of previously frozen tissue while maintaining the frozen state. This technique and the specialized equipment that is necessary to perform the technique is extremely useful for identifying specific molecules in cells and tissues using antibodies tagged with a label that can be seen under a transmission electron microscope. As a result, the location of the molecule of interest can be pin-pointed with very high accuracy within cells and subcellular compartments. By preserving the molecular components of cells and tissues by freezing, rather than with chemical fixatives, molecules of interest are less likely to be damaged beyond recognition by antibodies, so sensitivity of the labeling technique is greatly improved. Furthermore, the technique does not require the use of detergents to make cell membranes permeable to antibodies, a necessary step of conventional antibody-labeling procedures that drastically diminishes the quality of cellular ultrastructure. The research that will be facilitated by the requested equipment includes: 1) analysis of the effects of maternal stress on the developing nervous system of offspring, 2) study of neural circuits in the mammalian eye, 3) study of the defensive responses of poplar trees as induced by herbivore attacks, and 4) study of the developing nervous system in animals that have a larval stage in the life cycle. In addition, the equipment will be used to explore the use of very thin frozen sections for a mass spectrometry technique, uniquely available at the University of Victoria - Genome BC Proteomics facility in Victoria. This technique allows proteins to be identified and localized with high accuracy within cells and tissues and will be applied to a study of seed development in economically important conifers. A skilled technician employed full time in our Electron Microscopy Laboratory would provide expert training for graduate students in the method of cutting frozen, ultrathin sections.

该提案的五位共同申请人希望为我们现有的超薄切片机购买一个附件，该附件由NSERC于2003年资助，使我们能够在保持冷冻状态的同时切割非常薄的先前冷冻组织切片。该技术和执行该技术所需的专用设备对于使用标记有标签的抗体识别细胞和组织中的特定分子非常有用，该标签可以在透射电子显微镜下看到。因此，可以在细胞和亚细胞区室内以非常高的精度精确定位感兴趣分子的位置。通过冷冻保存细胞和组织的分子组分，而不是用化学固定剂，感兴趣的分子不太可能被破坏而无法被抗体识别，因此标记技术的灵敏度大大提高。此外，该技术不需要使用去污剂来使细胞膜对抗体可渗透，这是常规抗体标记程序的必要步骤，其大大降低了细胞超微结构的质量。所要求的设备将有助于进行的研究包括：1）分析母体压力对后代神经系统发育的影响，2）研究哺乳动物眼睛的神经回路，3）研究白杨受到草食动物攻击后的防御反应，4）研究生命周期中处于幼虫阶段的动物的神经系统发育。此外，该设备将用于探索使用非常薄的冷冻切片进行质谱分析技术，这在维多利亚大学-维多利亚的基因组BC蛋白质组学设施中是独一无二的。这种技术允许蛋白质被识别和定位在细胞和组织内的高精度，并将被应用于种子发育的研究在经济上重要的针叶树。我们的电子显微镜实验室全职雇用一名熟练的技术人员，为研究生提供冷冻切片切割方法的专家培训。