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.

doi.org/10.1038/nprot.2011.441, hdl.handle.net/1765/57127
Nature Protocols
Biophysical Genomics, Department Cell Biology & Genetics

Yokomizo, T., Yamada-Inagawa, T., Yzaguirre, A., Chen, M., Speck, N., & Dzierzak, E. (2012). Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos. Nature Protocols, 7(3), 421–423. doi:10.1038/nprot.2011.441