Optimizing tissue clearing and imaging methods for human brain tissue

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 (TM). 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 mu m and immunostaining depth exceeded 200 mu m in 4 days. Conclusions We present optimized sample preparation and staining protocols for the visualization of three-dimensional macrostructure in the human brain.

Automated summary

Fresh samples outperformed cadaver samples in terms of clearing efficiency, artifacts, and DAPI staining efficiency, but had similar GFAP staining efficiency. The penetration depth and DAPI staining in fresh samples improved with longer incubation periods. 4DS-C was the best method with the deepest penetration.