Subcellular Localization of Neuronal Proteins by Immunoelectron Microscopy

Open Access
Author:
Cui, Ruda
Area of Honors:
Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Richard W Ordway, Thesis Supervisor
  • Bernhard Luscher, Honors Advisor
Keywords:
  • immunocytochemistry
  • electron microscopy
  • Drosophila
  • nervous system
  • microscopy
Abstract:
Immuno-Electron Microscopy (IEM) combines ultrastructural analysis with immunocytochemistry to determine the distribution of proteins with nanometer resolution. IEM can provide new understanding of molecular basis of cellular processes by revealing spatial relationships of specific proteins and subcellular organelles. Such detailed information about the location of proteins, both under normal conditions and after a genetic, pharmacological or environmental perturbation, is of tremendous value in understanding the functions and interactions of specific proteins. Traditional immunocytochemistry and IEM methods utilize detergents to allow antibodies to enter cells to label specific intracellular proteins. However, this process destroys cellular membranes. While this damage in not problematic when imaging samples by light microscopy, it produces a marked deterioration of cellular ultrastructure as observed by high-resolution transmission electron microscopy. Our studies build on previous work to develop and optimize IEM methods for analysis of subcellular protein distribution within the nervous system of the Drosophila adult. This approach utilizes correlative light and electron microscopy to determine the distribution of specific proteins in the context of the whole organism. In addition, these methods eliminate the use of detergents for the immunocytochemistry aspect of IEM and permit both superior preservation of ultrastructure and effective labeling of intracellular proteins. My project is focused on further optimization and application of these methods for analysis of subcellular protein distributions at synapses within the brain and thoracic neural tissue of the Drosophila adult.