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Procedures for the quantification of whole-tissue immunofluorescence images obtained at single-cell resolution during murine tubular organ development.

Hirashima T, Adachi T - PLoS ONE (2015)

Bottom Line: However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed.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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

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.

No MeSH data available.


Whole-tissue fluorescence images of F-actin and E-cadherin.The spatial distribution of F-actin (left) and E-cadherin (right) during epididymal (upper), kidney (middle), and lung (bottom) development is shown. Each organ was dissected at E16.5 for epididymis and at E13.5 for both kidney and lung. Dotted orange lines in the middle row represent the tissue boundary of the epithelial renal tubule. For the images obtained with F-actin staining, a maximum intensity projection was applied over 30 μm with 2-μm intervals for the three organs. The E-cadherin images are single section views. Scale bar: 50 μm.
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pone.0135343.g001: Whole-tissue fluorescence images of F-actin and E-cadherin.The spatial distribution of F-actin (left) and E-cadherin (right) during epididymal (upper), kidney (middle), and lung (bottom) development is shown. Each organ was dissected at E16.5 for epididymis and at E13.5 for both kidney and lung. Dotted orange lines in the middle row represent the tissue boundary of the epithelial renal tubule. For the images obtained with F-actin staining, a maximum intensity projection was applied over 30 μm with 2-μm intervals for the three organs. The E-cadherin images are single section views. Scale bar: 50 μm.

Mentions: We first show images obtained via whole-tissue fluorescence labeling of F-actin and E-cadherin, both of which are generally expressed in epithelial cells, in the developing epididymis (E16.5), kidney (E13.5), and lung (E13.5) (Fig 1). Although these two proteins are known to be key factors for the generation of cellular physical force associated with maintaining epithelial integrity during tissue morphogenesis, the exact distribution of these proteins in whole internal organs remains elusive because the analysis has been mainly performed via slice sectioning [18–21].


Procedures for the quantification of whole-tissue immunofluorescence images obtained at single-cell resolution during murine tubular organ development.

Hirashima T, Adachi T - PLoS ONE (2015)

Whole-tissue fluorescence images of F-actin and E-cadherin.The spatial distribution of F-actin (left) and E-cadherin (right) during epididymal (upper), kidney (middle), and lung (bottom) development is shown. Each organ was dissected at E16.5 for epididymis and at E13.5 for both kidney and lung. Dotted orange lines in the middle row represent the tissue boundary of the epithelial renal tubule. For the images obtained with F-actin staining, a maximum intensity projection was applied over 30 μm with 2-μm intervals for the three organs. The E-cadherin images are single section views. Scale bar: 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4530862&req=5

pone.0135343.g001: Whole-tissue fluorescence images of F-actin and E-cadherin.The spatial distribution of F-actin (left) and E-cadherin (right) during epididymal (upper), kidney (middle), and lung (bottom) development is shown. Each organ was dissected at E16.5 for epididymis and at E13.5 for both kidney and lung. Dotted orange lines in the middle row represent the tissue boundary of the epithelial renal tubule. For the images obtained with F-actin staining, a maximum intensity projection was applied over 30 μm with 2-μm intervals for the three organs. The E-cadherin images are single section views. Scale bar: 50 μm.
Mentions: We first show images obtained via whole-tissue fluorescence labeling of F-actin and E-cadherin, both of which are generally expressed in epithelial cells, in the developing epididymis (E16.5), kidney (E13.5), and lung (E13.5) (Fig 1). Although these two proteins are known to be key factors for the generation of cellular physical force associated with maintaining epithelial integrity during tissue morphogenesis, the exact distribution of these proteins in whole internal organs remains elusive because the analysis has been mainly performed via slice sectioning [18–21].

Bottom Line: However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed.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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

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.

No MeSH data available.