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 journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)421-31
Number of pages11
JournalNature Protocols
Volume7
Issue number3
DOIs
Publication statusPublished - Mar 2012

Keywords

  • 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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