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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™. Clearing efficiency and artifact levels were calcu...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
SAGE Publications
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8166401/ https://www.ncbi.nlm.nih.gov/pubmed/33771067 http://dx.doi.org/10.1177/03000605211001729 |
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author | Kim, Min Sun Ahn, Jang Ho Mo, Ji Eun Song, Ha Young Cheon, Deokhyeon Yoo, Seong Ho Choi, Hyung Jin |
author_facet | Kim, Min Sun Ahn, Jang Ho Mo, Ji Eun Song, Ha Young Cheon, Deokhyeon Yoo, Seong Ho Choi, Hyung Jin |
author_sort | Kim, Min Sun |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8166401 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | SAGE Publications |
record_format | MEDLINE/PubMed |
spelling | pubmed-81664012021-06-07 Optimizing tissue clearing and imaging methods for human brain tissue Kim, Min Sun Ahn, Jang Ho Mo, Ji Eun Song, Ha Young Cheon, Deokhyeon Yoo, Seong Ho Choi, Hyung Jin J Int Med Res Pre-Clinical Research Report 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. SAGE Publications 2021-03-26 /pmc/articles/PMC8166401/ /pubmed/33771067 http://dx.doi.org/10.1177/03000605211001729 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by-nc/4.0/Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). |
spellingShingle | Pre-Clinical Research Report Kim, Min Sun Ahn, Jang Ho Mo, Ji Eun Song, Ha Young Cheon, Deokhyeon Yoo, Seong Ho Choi, Hyung Jin Optimizing tissue clearing and imaging methods for human brain tissue |
title | Optimizing tissue clearing and imaging methods for human brain tissue |
title_full | Optimizing tissue clearing and imaging methods for human brain tissue |
title_fullStr | Optimizing tissue clearing and imaging methods for human brain tissue |
title_full_unstemmed | Optimizing tissue clearing and imaging methods for human brain tissue |
title_short | Optimizing tissue clearing and imaging methods for human brain tissue |
title_sort | optimizing tissue clearing and imaging methods for human brain tissue |
topic | Pre-Clinical Research Report |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8166401/ https://www.ncbi.nlm.nih.gov/pubmed/33771067 http://dx.doi.org/10.1177/03000605211001729 |
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