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. 2012 Feb 9;7(3):421-31.
doi: 10.1038/nprot.2011.441.

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

Tomomasa Yokomizo  1 , Tomoko Yamada-Inagawa, Amanda D Yzaguirre, Michael J Chen, Nancy A Speck, Elaine Dzierzak
Affiliations

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

Tomomasa Yokomizo et al. Nat Protoc. .

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.

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Figures

Figure 1
Figure 1
Representative overview of the protocol for two-color staining.
Figure 2
Figure 2
Increasing tissue transparency using BABB solution. (a) Appearance of E10.5 mouse embryo (before fixation). (b,c) Boxed region was scanned after transferring immunostained embryos into PBS (b) or BABB (c). (b) Although maximum laser power was used, a clear structure was not observed in the embryo immersed in PBS. (c) In contrast, the dorsal aorta and hematopoietic clusters were clearly observed in the embryo immersed in BABB. Scale bars, 100 µm. Arrows indicate ventral (V) and dorsal (D) walls of dorsal aorta. Yellow arrowheads indicate hematopoietic clusters.
Figure 3
Figure 3
Embryo trimming. (a–c) Schematic diagram of embryo trimming. First, remove the head (i in a). To remove the lateral body wall (iii in a, left, and red dashed lines in b), an incision is made along the red line (iii in a, right). E10.5 embryo pictures before and after removing the lateral body wall are shown in b and c, respectively. Mesentery (blue arrows) can be seen after removing the lateral body wall. BC, body cavity; DA, dorsal aorta; NT, neural tube; UA, umbilical artery. Scale bars, 500 µm.
Figure 4
Figure 4
Slide preparation. (a) A slide with a FastWell. (b) Diagram representing a side view of the completed slide. See Steps 20–35 for preparation.
Figure 5
Figure 5
Three-dimensional reconstruction of the dorsal aorta region stained with c-Kit (green)- and CD31 (magenta)-specific antibodies at E10.5 (35 somite pairs). (a) Representative raw images (xy confocal sections) of the sample. (b) 3D reconstructed image. Scale bars, 100 µm.
Figure 6
Figure 6
Quantification procedure of c-Kit+ hematopoietic cluster cells in the dorsal aorta. (a) First, a 3D reconstructed image is made from serial xy section images. (b–d) After splitting into three channels (magenta, green and blue), the number of c-Kit+ cells (green) is counted. (e) Magnification of the boxed region in c. As c-Kit is a cell surface marker, the border of cells within clusters is clearly distinct. (f) The number of circulating c-Kit+ cells (i.e., cells that do not attach to the wall of dorsal aorta) is counted using an orthogonal view. Move the position of the yz plane (red line in xy data) manually, and identify circulating c-Kit+ cells in yz data (white arrowhead). To obtain the number of c-Kit+ hematopoietic cluster cells, the number of circulating c-Kit+ cells is subtracted from the number of c-Kit+ cells in image c. To cover the whole dorsal aorta, six or seven scans are required. Scale bars, 100 µm.
Figure 7
Figure 7
Three-dimensional reconstructed pictures at low and high magnifications. (a) E10.5 embryo stained with anti-CD31 antibody. The picture was taken with a ×10 objective (EC Plan-Neofluar ×10/NA 0.3). Blood vessels of various sizes throughout the embryo are observed. DA, dorsal aorta; UA, umbilical artery; VA, vitelline artery. (b) E10.5 embryo stained with anti–c-Kit and anti-CD31 antibodies. The picture was taken with a ×63 objective (Achroplan ×63/NA 0.95 W). c-Kit+ hematopoietic clusters attached to the CD31+ endothelial layer are observed.
Figure 8
Figure 8
Three-dimensional reconstructed pictures showing nuclear expression of phosphohistone H3. (a) E10.5 embryo stained with anti-phosphohistone H3 antibody (blue) and anti-CD31 antibody (magenta). The picture was taken with a ×10 objective. Composite image of 83 slices (optical sections of 163.54 µm thickness) is shown. (b) Image of the umbilical artery (boxed region of panel a) showing a large hematopoietic cluster with two cells expressing phosphohistone H3 (arrowheads), taken with a ×20 objective (zoom 3.28). DA, dorsal aorta; UA, umbilical artery. Scale bar, 300 µm.
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