DiGiovanni Jerome, Sun Tao, Sheng Zu-Hang
National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA.
Curr Protoc Neurosci. 2012 Apr;Chapter 2:Unit 2.7.1-22. doi: 10.1002/0471142301.ns0207s59.
Cloning and characterization of synaptic vesicle proteins and their binding counterparts on the presynaptic plasma membrane have greatly advanced our understanding of the molecular mechanisms involved in the synaptic vesicle cycle and neurotransmitter release. This unit discusses multidisciplinary approaches to characterize proteins from synaptosome-enriched subcellular fractions and localize them within cultured neurons. The first approach regroups methods used to isolate synaptic vesicles from rat brain synaptosomal preparations, allowing for specific biochemical investigation of synaptic vesicle proteins. The second is a detailed procedure for pre-embedding immunogold staining and electron microscopic observation, which permits the morphological identification of proteins in individual vesicles at intact synapses. Additionally, this chapter proposes methods for light microscopic examination of hippocampal neurons. It includes procedures for embryonic and postnatal hippocampal neuron culture and describes an immunocytochemical staining protocol used to investigate synaptic vesicle protein localization with respect to other proteins or subcellular structures.
突触小泡蛋白及其在突触前质膜上的结合对应物的克隆和特性分析极大地推动了我们对突触小泡循环和神经递质释放所涉及分子机制的理解。本单元讨论了多学科方法，用于鉴定富含突触体的亚细胞组分中的蛋白质，并将它们定位在培养的神经元内。第一种方法重新整理了从大鼠脑突触体制备物中分离突触小泡的方法，以便对突触小泡蛋白进行特定的生化研究。第二种方法是预包埋免疫金染色和电子显微镜观察的详细步骤，它能够在完整突触处对单个小泡中的蛋白质进行形态学鉴定。此外，本章还提出了海马神经元光学显微镜检查的方法。它包括胚胎期和出生后海马神经元培养的步骤，并描述了一种免疫细胞化学染色方案，用于研究突触小泡蛋白相对于其他蛋白质或亚细胞结构的定位。
