Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos.
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.
Department of Cell Biology, Erasmus Medical Center Stem Cell Institute, Erasmus Medical Center, Rotterdam, The Netherlands. csity@nus.edu.sg
Tomomasa
T
Yokomizo
Tomoko
T
Yamada-Inagawa
Amanda D
AD
Yzaguirre
Michael J
MJ
Chen
Nancy A
NA
Speck
Elaine
E
Dzierzak
D000818:Animals; D001011:Aorta / Q000166:cytology*; D004622:Embryo, Mammalian / Q000166:cytology*; D005453:Fluorescence; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D016707:Tissue Fixation / Q000379:methods