Labeling and Grafting of Cells

1. Place a 50-|L drop of medium (the manipulation drop) slightly off-center in the lid of a Petri dish (Note 19). Place another drop of medium (for holding cells for grafting) or a drop of carbocyanine dye (for labeling of embryos) next to the first drop (Fig. 4B).

2. Fill the Petri dish with light paraffin oil to cover the drops.

3. Transfer embryos from the culture to the manipulation drop in the Petri dish by mouth pipeting.

4. Angled pipets are used for holding and labeling the embryos. Place the pipets in the instrument holders of the manipulator. Tilt the manipulators so that the tips of the pipets are immersed in the paraffin oil and are pointing to the bottom of the Petri dish. Figure 6 shows the setup for manipulating 7.5-d embryos.

Fig. 8. Grafting of cells to a 6.5-d embryo. The embryo is held on the anterior side opposite the intended site of grafting by the holding pipet (h). The cells are grafted into the epiblast (ep) on the posterior side of the embryo (*marks the position of the primitive streak) immediately proximal to the margin of the distal cap. Bar = 20 |im.

5. Focus the microscope on the embryos in the manipulation drop. Lower the pipets into the drop. Manipulation is carried out according to Subheading 3.3.2., steps 7-20 and Subheading 3.3.3., steps 3-8 and Note 20.

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