优化人脑组织的组织透明化和成像方法。

Optimizing tissue clearing and imaging methods for human brain tissue.

机构信息

Functional Neuroanatomy of Metabolism Regulation Laboratory, Department of Anatomy, Seoul National University College of Medicine, Seoul, South Korea.

Institute of Forensic Medicine and Department of Forensic Medicine, Seoul National University College of Medicine, Seoul, South Korea.

出版信息

J Int Med Res. 2021 Mar;49(3):3000605211001729. doi: 10.1177/03000605211001729.

DOI:10.1177/03000605211001729

PMID:33771067

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8166401/

Abstract

OBJECTIVES

To identify optimum sample conditions for human brains, we compared the clearing efficiency, antibody staining efficiency, and artifacts between fresh and cadaver samples.

METHODS

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.

RESULTS

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.

CONCLUSIONS

We present optimized sample preparation and staining protocols for the visualization of three-dimensional macrostructure in the human brain.

摘要

目的

为了确定适用于人脑的最佳样本条件，我们比较了新鲜和尸检样本的清除效率、抗体染色效率和伪影。

方法

使用 X-CLARITY™对新鲜和尸检样本进行清除。使用 ImageJ 计算清除效率和伪影水平，并在共聚焦显微镜成像后评估抗体染色效率。比较了三种染色方法：4 天染色（4DS）、11 天染色（11DS）和 4 天商业化试剂盒染色（4DS-C）。然后通过评估染色时间、深度、方法复杂性、污染和成本来选择最佳染色方法。

结果

新鲜样本在清除时间和质量、伪影和 4'，6-二脒基-2-苯基吲哚（DAPI）染色效率方面优于尸检样本，但神经胶质纤维酸性蛋白（GFAP）染色效率与尸检样本相似。随着孵育时间的延长，新鲜样本的穿透深度和 DAPI 染色得到改善。4DS-C 是最好的方法，具有最深的穿透性。可以三维可视化包含血管、细胞核和星形胶质细胞的人脑图像。化学染料染色深度在 4 天内达到 800μm，免疫染色深度超过 200μm。