Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos

Tomomasa Yokomizo; Tomoko Yamada-Inagawa; Amanda D Yzaguirre; Michael J Chen; Nancy A Speck; Elaine Dzierzak

doi:10.1038/nprot.2011.441

Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos

Tomomasa Yokomizo, Tomoko Yamada-Inagawa, Amanda D Yzaguirre, Michael J Chen, Nancy A Speck, Elaine Dzierzak

Research output: Contribution to journal › Article › peer-review

Abstract / Description of output

We describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development and 3D architecture of other tissues. Previously, direct whole-mount imaging has been limited to external tissue layers owing to poor laser penetration of dense, opaque tissue. Our whole-embryo imaging method enables detailed quantitative and qualitative analysis of cells within the dorsal aorta of embryonic day (E) 10.5-11.5 embryos after the removal of only the head and body walls. In this protocol we describe the whole-mount fixation and multimarker staining procedure, the tissue transparency treatment, microscopy and the analysis of resulting images. A typical two-color staining experiment can be performed and analyzed in ∼6 d.

Original language	English
Pages (from-to)	421-31
Number of pages	11
Journal	Nature Protocols
Volume	7
Issue number	3
DOIs	https://doi.org/10.1038/nprot.2011.441
Publication status	Published - Mar 2012

Keywords / Materials (for Non-textual outputs)

Animals
Aorta
Embryo, Mammalian
Fluorescence
Hematopoietic Stem Cells
Image Processing, Computer-Assisted
Immunohistochemistry
Mice
Microscopy, Confocal
Staining and Labeling
Tissue Fixation

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10.1038/nprot.2011.441

Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryosAccepted author manuscript, 5.14 MB

http://www.nature.com/nprot/journal/v7/n3/full/nprot.2011.441.html

Cite this

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title = "Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos",

abstract = "We describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development and 3D architecture of other tissues. Previously, direct whole-mount imaging has been limited to external tissue layers owing to poor laser penetration of dense, opaque tissue. Our whole-embryo imaging method enables detailed quantitative and qualitative analysis of cells within the dorsal aorta of embryonic day (E) 10.5-11.5 embryos after the removal of only the head and body walls. In this protocol we describe the whole-mount fixation and multimarker staining procedure, the tissue transparency treatment, microscopy and the analysis of resulting images. A typical two-color staining experiment can be performed and analyzed in ∼6 d.",

keywords = "Animals, Aorta, Embryo, Mammalian, Fluorescence, Hematopoietic Stem Cells, Image Processing, Computer-Assisted, Immunohistochemistry, Mice, Microscopy, Confocal, Staining and Labeling, Tissue Fixation",

author = "Tomomasa Yokomizo and Tomoko Yamada-Inagawa and Yzaguirre, {Amanda D} and Chen, {Michael J} and Speck, {Nancy A} and Elaine Dzierzak",

year = "2012",

month = mar,

doi = "10.1038/nprot.2011.441",

language = "English",

volume = "7",

pages = "421--31",

journal = "Nature Protocols",

issn = "1754-2189",

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T1 - Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos

AU - Yokomizo, Tomomasa

AU - Yamada-Inagawa, Tomoko

AU - Yzaguirre, Amanda D

AU - Chen, Michael J

AU - Speck, Nancy A

AU - Dzierzak, Elaine

PY - 2012/3

Y1 - 2012/3

N2 - We describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development and 3D architecture of other tissues. Previously, direct whole-mount imaging has been limited to external tissue layers owing to poor laser penetration of dense, opaque tissue. Our whole-embryo imaging method enables detailed quantitative and qualitative analysis of cells within the dorsal aorta of embryonic day (E) 10.5-11.5 embryos after the removal of only the head and body walls. In this protocol we describe the whole-mount fixation and multimarker staining procedure, the tissue transparency treatment, microscopy and the analysis of resulting images. A typical two-color staining experiment can be performed and analyzed in ∼6 d.

AB - We describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development and 3D architecture of other tissues. Previously, direct whole-mount imaging has been limited to external tissue layers owing to poor laser penetration of dense, opaque tissue. Our whole-embryo imaging method enables detailed quantitative and qualitative analysis of cells within the dorsal aorta of embryonic day (E) 10.5-11.5 embryos after the removal of only the head and body walls. In this protocol we describe the whole-mount fixation and multimarker staining procedure, the tissue transparency treatment, microscopy and the analysis of resulting images. A typical two-color staining experiment can be performed and analyzed in ∼6 d.

KW - Animals

KW - Aorta

KW - Embryo, Mammalian

KW - Fluorescence

KW - Hematopoietic Stem Cells

KW - Image Processing, Computer-Assisted

KW - Immunohistochemistry

KW - Mice

KW - Microscopy, Confocal

KW - Staining and Labeling

KW - Tissue Fixation

U2 - 10.1038/nprot.2011.441

DO - 10.1038/nprot.2011.441

M3 - Article

C2 - 22322215

SN - 1754-2189

VL - 7

SP - 421

EP - 431

JO - Nature Protocols

JF - Nature Protocols

IS - 3

ER -

Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos

Abstract / Description of output

Keywords / Materials (for Non-textual outputs)

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