Optimizing tissue clearing and imaging methods for human brain tissue.
Optimizing tissue clearing and imaging methods for human brain tissue.
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DOI：
10.1177/03000605211001729
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发表时间：
2021-03
期刊：
影响因子：
--
通讯作者：
Choi HJ
中科院分区：
文献类型：
--
作者：
Kim MS;Ahn JH;Mo JE;Song HY;Cheon D;Yoo SH;Choi HJ
To identify optimum sample conditions for human brains, we compared the clearing efficiency, antibody staining efficiency, and artifacts between fresh and cadaver samples. Fresh and cadaver samples were cleared using X-CLARITY™. Clearing efficiency and artifact levels were calculated using ImageJ, and antibody staining efficiency was evaluated after confocal microscopy imaging. Three staining methods were compared: 4-day staining (4DS), 11-day staining (11DS), and 4-day staining with a commercial kit (4DS-C). The optimum staining method was then selected by evaluating staining time, depth, method complexity, contamination, and cost. Fresh samples outperformed cadaver samples in terms of the time and quality of clearing, artifacts, and 4′,6-diamidino-2-phenylindole (DAPI) staining efficiency, but had a glial fibrillary acidic protein (GFAP) staining efficiency that was similar to that of cadaver samples. The penetration depth and DAPI staining improved in fresh samples as the incubation period lengthened. 4DS-C was the best method, with the deepest penetration. Human brain images containing blood vessels, cell nuclei, and astrocytes were visualized three-dimensionally. The chemical dye staining depth reached 800 µm and immunostaining depth exceeded 200 µm in 4 days. We present optimized sample preparation and staining protocols for the visualization of three-dimensional macrostructure in the human brain.
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