Shan Qing-Hong, Qin Xin-Ya, Zhou Jiang-Ning
Chinese Academy of Science Key Laboratory of Brain Function and Diseases, Division of Life Sciences and Medicine, School of Life Sciences, University of Science and Technology of China, Hefei 230026, China.
Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China.
Int J Mol Sci. 2022 Mar 25;23(7):3575. doi: 10.3390/ijms23073575.
Obtaining fine neuron morphology and connections data is extraordinarily useful in understanding the brain's functionality. Golgi staining is a widely used method for revealing neuronal morphology. However, Golgi-Cox-stained tissue is difficult to image in three dimensions and lacks cell-type specificity, limiting its use in neuronal circuit studies. Here, we describe an expansion-based method for rapidly clearing Golgi-Cox-stained tissue. The results show that 1 mm thick Golgi-Cox-stained tissue can be cleared within 6 hours with a well preserved Golgi-Cox-stained signal. At the same time, we found for the first time that the cleared Golgi-Cox-stained samples were compatible with three-dimensional (3D) immunostaining and multi-round immunostaining. By combining the Golgi-Cox staining with tissue clearing and immunostaining, Golgi-Cox-stained tissue could be used for large-volume 3D imaging, identification of cell types of Golgi-Cox-stained cells, and reconstruction of the neural circuits at dendritic spines level. More importantly, these methods could also be applied to samples from human brains, providing a tool for analyzing the neuronal circuit of the human brain.
获取精细的神经元形态和连接数据对于理解大脑功能非常有用。高尔基染色是一种广泛用于揭示神经元形态的方法。然而，高尔基-考克斯染色的组织难以进行三维成像且缺乏细胞类型特异性，限制了其在神经元回路研究中的应用。在此，我们描述了一种基于膨胀的方法，用于快速清除高尔基-考克斯染色的组织。结果表明，1毫米厚的高尔基-考克斯染色组织可在6小时内被清除，且高尔基-考克斯染色信号保存良好。同时，我们首次发现清除后的高尔基-考克斯染色样本与三维（3D）免疫染色和多轮免疫染色兼容。通过将高尔基-考克斯染色与组织清除和免疫染色相结合，高尔基-考克斯染色的组织可用于大体积3D成像、鉴定高尔基-考克斯染色细胞的细胞类型以及在树突棘水平重建神经回路。更重要的是，这些方法也可应用于来自人类大脑的样本，为分析人类大脑的神经元回路提供了一种工具。
