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ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabe...

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Autores principales: Lee, Eunsoo, Sun, Woong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409193/
https://www.ncbi.nlm.nih.gov/pubmed/28117772
http://dx.doi.org/10.3791/54904
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author Lee, Eunsoo
Sun, Woong
author_facet Lee, Eunsoo
Sun, Woong
author_sort Lee, Eunsoo
collection PubMed
description The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique–pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.
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spelling pubmed-54091932017-05-12 ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging Lee, Eunsoo Sun, Woong J Vis Exp Bioengineering The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique–pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses. MyJove Corporation 2016-12-31 /pmc/articles/PMC5409193/ /pubmed/28117772 http://dx.doi.org/10.3791/54904 Text en Copyright © 2016, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Bioengineering
Lee, Eunsoo
Sun, Woong
ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging
title ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging
title_full ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging
title_fullStr ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging
title_full_unstemmed ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging
title_short ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging
title_sort act-presto: biological tissue clearing and immunolabeling methods for volume imaging
topic Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409193/
https://www.ncbi.nlm.nih.gov/pubmed/28117772
http://dx.doi.org/10.3791/54904
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