背景:组织清除通过增加组织的透明度来实现各种组织的深度成像,但是对整个大脑等大体积组织的免疫染色存在局限性。
方法:这里,我们使用一种称为高速清除和高分辨率染色(HCHS)的新型清除技术清除并对整个小鼠脑组织进行免疫染色。我们使用共聚焦显微镜和光片荧光显微镜(LSFM)观察了清除的大脑中的神经结构。使用计算重建算法分析重建的3D图像。
结果:在来自Gad-绿色荧光蛋白(GFP)小鼠和Thy1-黄色荧光蛋白(YFP)小鼠的清除脑的三维(3D)图像中良好地观察到各种神经结构。在HCHS之后,两个转基因小鼠的固有荧光信号被保留。此外,大脑的大规模3D成像,使用温和的基于洗涤剂的溶液通过HCHS方法进行免疫染色,允许对几种神经元标记如c-Fos进行全局拓扑分析,神经元核蛋白(NeuN),微管相关蛋白2(Map2),Tuj1,胶质纤维酸性蛋白(GFAP),和酪氨酸羟化酶(TH)在整个小鼠脑组织的各个解剖区域。最后,通过与各种现有的组织清除方法的比较,如CUBIC,Visikol,和3DISCO,证实了HCHS方法导致相对较少的组织变形和较高的荧光保留。
结论:结论:基于新型组织清除技术(HCHS)的3D成像技术的发展将能够对大脑中存在的神经和血管网络进行详细的空间分析。
BACKGROUND: Tissue clearing enables deep imaging in various tissues by increasing the transparency of tissues, but there were limitations of immunostaining of the large-volume tissues such as the whole brain.
METHODS: Here, we cleared and immune-stained whole mouse brain tissues using a novel clearing technique termed high-speed clearing and high-resolution staining (HCHS). We observed neural structures within the cleared brains using both a confocal microscope and a light-sheet fluorescence microscope (LSFM). The reconstructed 3D images were analyzed using a computational reconstruction algorithm.
RESULTS: Various neural structures were well observed in three-dimensional (3D) images of the cleared brains from Gad-green fluorescent protein (GFP) mice and Thy 1-yellow fluorescent protein (YFP) mice. The intrinsic fluorescence signals of both transgenic mice were preserved after HCHS. In addition, large-scale 3D imaging of brains, immune-stained by the HCHS method using a mild detergent-based solution, allowed for the global topological analysis of several neuronal markers such as c-Fos, neuronal nuclear protein (NeuN), Microtubule-associated protein 2 (Map2), Tuj1, glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) in various anatomical regions in the whole mouse brain tissues. Finally, through comparisons with various existing tissue clearing methodologies such as CUBIC, Visikol, and 3DISCO, it was confirmed that the HCHS methodology results in relatively less tissue deformation and higher fluorescence retention.
CONCLUSIONS: In conclusion, the development of 3D imaging based on novel tissue-clearing techniques (HCHS) will enable detailed spatial analysis of neural and vascular networks present within the brain.