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Title: Procedures for the quantification of whole-tissue immunofluorescence images obtained at single-cell resolution during murine tubular organ development.
Authors: Hirashima, Tsuyoshi  kyouindb  KAKEN_id  orcid https://orcid.org/0000-0001-7323-9627 (unconfirmed)
Adachi, Taiji  kyouindb  KAKEN_id  orcid https://orcid.org/0000-0001-5280-4156 (unconfirmed)
Author's alias: 平島, 剛志
Issue Date: 10-Aug-2015
Publisher: Public Library of Science
Journal title: PLOS ONE
Volume: 10
Issue: 8
Thesis number: e0135343
Abstract: Whole-tissue quantification at single-cell resolution has become an inevitable approach for further quantitative understanding of morphogenesis in organ development. The feasibility of the approach has been dramatically increased by recent technological improvements in optical tissue clearing and microscopy. However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed. To provide the appropriate procedure, we here show tips for each critical step of the entire process, including fixation for immunofluorescence, optical clearing, and digital image processing, using developing murine internal organs such as epididymis, kidney, and lung as an example. Through comparison of fixative solutions and of clearing methods, we found optimal conditions to achieve clearer deep-tissue imaging of specific immunolabeled targets and explain what methods result in vivid volume imaging. In addition, we demonstrated that three-dimensional digital image processing after optical clearing produces objective quantitative data for the whole-tissue analysis, focusing on the spatial distribution of mitotic cells in the epididymal tubule. The procedure for the whole-tissue quantification shown in this article should contribute to systematic measurements of cellular processes in developing organs, accelerating the further understanding of morphogenesis at the single cell level.
Rights: © 2015 Hirashima, Adachi. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
URI: http://hdl.handle.net/2433/202559
DOI(Published Version): 10.1371/journal.pone.0135343
PubMed ID: 26258587
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