Investigation of synaptic vesicle membrane proteins using multidisciplinary approaches, particularly to characterize synaptic vesicle proteins in synapses, can greatly advance our knowledge of the molecular mechanisms involved in synaptic vesicle exocytosis and neurotransmission. Three approaches are presented in this unit to identify and characterize synaptic vesicle proteins. The first is a subcellular fractionation method used to isolate synaptic vesicles from rat brain synaptosomal preparations, which can then be used in a variety of biochemical studies on synaptic vesicle proteins. The second is a detailed procedure for pre-embedding immunogold staining and electron microscopic observation, techniques that permit the morphological identification of synaptic vesicle proteins in individual vesicles at the intact synapse. Finally, a protocol for immunocytochemical staining of cultured hippocampal neurons for light microscopic examination is provided, which allows one to stain multisynaptic vesicle proteins and determine their localization in relation to other proteins or subcellular structures in synapses.

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