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

该提案的五名共同申请人希望为我们现有的超微切片机购买附件，该设备于2003年由NSERC资助，使我们能够在保持冷冻状态的情况下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带有标签的抗体识别细胞和组织中的特定分子非常有用，这种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中以非常高的精度精确定位感兴趣的分子的位置。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，目标分子不太可能被抗体破坏而无法识别，因此大大提高了标记技术的灵敏度。此外，该技术不需要使用洗涤剂使细胞膜对抗体渗透，这是传统抗体标记程序的必要步骤，会大大降低细胞超微结构的质量。所要求的设备将促进的研究包括：1)母性应激对后代发育中的神经系统的影响的分析，2)哺乳动物眼睛神经回路的研究，3)草食动物攻击诱导的杨树防御反应的研究，以及4)生命周期中处于幼虫阶段的动物神经系统发育的研究。此外，该设备将用于探索使用非常薄的冷冻切片进行质谱技术，这是维多利亚大学基因组BC蛋白质组学设施独有的。这项技术可以在细胞和组织内高精度地识别和定位蛋白质，并将应用于经济上重要的针叶树种子发育的研究。我们的电子显微镜实验室将全职聘请一名技术熟练的技术人员，为研究生提供冷冻超薄切片切割方法的专业培训。

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.