Tremblay Marie-Eve, Riad Mustapha, Majewska Ania
Department of Neurobiology and Anatomy, University of Rochester, NY, USA.
J Vis Exp. 2010 Jul 20(41):2021. doi: 10.3791/2021.
Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.
透射电子显微镜（TEM）对于在高空间分辨率下观察中枢神经系统中的小胶质细胞、少突胶质细胞、星形胶质细胞和神经元亚细胞区室（树突、树突棘、轴突、轴突终末、胞体）及其细胞内细胞器和细胞骨架极为有用。结合TEM，包埋前免疫细胞化学能够区分特征和识别标准较少的细胞成分（例如，使用针对小胶质细胞特异性标志物Iba1（离子钙结合衔接分子1；如此处所示）的抗体时，小胶质细胞胞体和突起），鉴定细胞成分的神经递质含量（例如，血清素能）及其可溶性或膜结合蛋白的超微结构定位（例如，5-HT1A和EphA4受体）。在此，我们描述了一种用丙烯醛固定剂对小鼠进行经心灌注、脑移除和切片、免疫过氧化物酶-二氨基联苯胺（DAB）染色、树脂包埋以及脑切片超薄切片的方案。完成这些程序后，免疫染色材料即可用于TEM检查。如果严格执行，该技术在最佳超微结构保存和免疫细胞化学检测之间提供了绝佳的平衡。
