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.

这项提案的五位共同申请者希望为我们现有的超微切割机购买附件，该切割机于2003年由NSERC资助，使我们能够在保持冷冻状态的同时切割以前冻结的非常薄的组织切片。这项技术和实施这项技术所需的专门设备对于使用在透射电子显微镜下可以看到的标记的抗体来识别细胞和组织中的特定分子非常有用。因此，目标分子的位置可以在细胞和亚细胞隔间内以非常高的精确度被准确地定位。通过冷冻保存细胞和组织的分子成分，而不是使用化学固定剂，感兴趣的分子不太可能被抗体破坏到无法识别的程度，因此标记技术的灵敏度大大提高。此外，这项技术不需要使用洗涤剂来使细胞膜对抗体具有渗透性，这是传统抗体标记程序的必要步骤，大大降低了细胞超微结构的质量。所需设备将促进的研究包括：1)分析母体应激对后代发育神经系统的影响，2)哺乳动物眼睛中的神经回路研究，3)食草动物攻击诱导的杨树防御反应研究，以及4)生命周期中处于幼虫阶段的动物的神经系统发育研究。此外，该设备还将用于探索将超薄冰冻切片用于质谱学技术，该技术在维多利亚大学-基因组BC蛋白质组学设施中独一无二。这项技术使蛋白质能够在细胞和组织内高精度地识别和定位，并将被应用于经济上重要的针叶树种子发育的研究。一名全职受雇于我们电子显微镜实验室的熟练技术人员将为研究生提供冰冻超薄切片切割方法的专家培训。