Optimizing tissue clearing and imaging methods for human brain tissue

Min Sun Kim, Jang Ho Ahn, Ji Eun Mo, Ha Young Song, Deokhyeon Cheon, Seong Ho Yoo, Hyung Jin Choi

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
JournalJournal of International Medical Research
Issue number3
StatePublished - 2021


  • DAPI staining
  • GFAP staining
  • Tissue clearing
  • confocal microscopy
  • human brain

Fingerprint Dive into the research topics of 'Optimizing tissue clearing and imaging methods for human brain tissue'. Together they form a unique fingerprint.

Cite this