Bhattacharya Ushashi, Jhou Jia-Fong, Zou Yi-Fong, Abrigo Gerald, Lin Shu-Wei, Chen Yun-Hsuan, Chien Fan-Ching, Tai Hwan-Ching
Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan.
Department of Optics and Photonics, National Central University, Taoyuan, Taiwan.
Sci Rep. 2021 Sep 20;11(1):18583. doi: 10.1038/s41598-021-98142-1.
Synaptosomes are subcellular fractions prepared from brain tissues that are enriched in synaptic terminals, widely used for the study of neural transmission and synaptic dysfunction. Immunofluorescence imaging is increasingly applied to synaptosomes to investigate protein localization. However, conventional methods for imaging synaptosomes over glass coverslips suffer from formaldehyde-induced aggregation. Here, we developed a facile strategy to capture and image synaptosomes without aggregation artefacts. First, ethylene glycol bis(succinimidyl succinate) (EGS) is chosen as the chemical fixative to replace formaldehyde. EGS/glycine treatment makes the zeta potential of synaptosomes more negative. Second, we modified glass coverslips with 3-aminopropyltriethoxysilane (APTES) to impart positive charges. EGS-fixed synaptosomes spontaneously attach to modified glasses via electrostatic attraction while maintaining good dispersion. Individual synaptic terminals are imaged by conventional fluorescence microscopy or by super-resolution techniques such as direct stochastic optical reconstruction microscopy (dSTORM). We examined tau protein by two-color and three-color dSTORM to understand its spatial distribution within mouse cortical synapses, observing tau colocalization with synaptic vesicles as well postsynaptic densities.
突触体是从富含突触终末的脑组织中制备的亚细胞组分，广泛用于神经传递和突触功能障碍的研究。免疫荧光成像越来越多地应用于突触体以研究蛋白质定位。然而，在玻璃盖玻片上对突触体进行成像的传统方法存在甲醛诱导的聚集问题。在此，我们开发了一种简便的策略来捕获和成像突触体而不会产生聚集假象。首先，选择乙二醇双琥珀酰亚胺琥珀酸酯（EGS）作为化学固定剂来替代甲醛。EGS/甘氨酸处理使突触体的zeta电位更负。其次，我们用3-氨丙基三乙氧基硅烷（APTES）修饰玻璃盖玻片以赋予正电荷。EGS固定的突触体通过静电吸引自发地附着在修饰过的玻璃上，同时保持良好的分散性。通过传统荧光显微镜或直接随机光学重建显微镜（dSTORM）等超分辨率技术对单个突触终末进行成像。我们通过双色和三色dSTORM检测tau蛋白，以了解其在小鼠皮质突触内的空间分布，观察到tau与突触小泡以及突触后致密物共定位。
