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

点击翻译按钮获取中文摘要

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.