期刊
NATURE PROTOCOLS
卷 7, 期 3, 页码 421-431出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2011.441
关键词
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资金
- US National Institutes of Health [R37DK054077, R01HL091724]
- Netherlands BSIK Innovation Program [03040]
- European Science Foundation Program EuroSTELLS [01-011]
- Netherlands Genomics Initiative-Cancer Genomic Distinguished Scientist Award
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 similar to 6 d.
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