C

Fig. 4. Making grafting pipets. A glass bead is attached to the tip of the heating filament of the microforge. The bead can be heated and cooled instantly by switching on and off the current of the heating filament. The current is adjusted to yield optimal heating that is appropriate to the thickness and the thermal property of the glass capillary. (A,B) The heated glass bead contacts the shaft of the pipet at the position that will give the desired internal diameter. (C) Melting of the capillary wall into the glass bead. (D) Resultant break in the pipet after the bead cools down and retracts when the current is switched off. This produces a pipet of the desired internal diameter and an even tip.

2. Break off the tip of the injection pipet by touching it against the cold glass bead on the filament of a microforge. This will produce a pipet for dye labeling (Fig. 1B).

3. In order to make grafting pipets, pull the glass capillary as in step 1. Determine a position on the shaft of the pipet by measuring with a ocular micrometer on the eyepiece of the microscope of the microforge, where the glass capillary has an inner diameter appropriate for the size of the cell clumps . Break the shaft of the pipet (Fig. 4) by (1) bringing the pipet to the heated glass bead on the microforge filament so that the site of intended break just touches the bead, (2) as the capillary begins to fuse with the glass bead, turn off the power supply to the filament to cool down the filament instantly. The retraction of the filament as it cools down will snap the pipet precisely at where the capillary fuses to the bead.

4. Beveled pipet (Fig. 3D) are made from the grafting pipet made in step 3 (Fig. 3C) (see Notes 9 and 10). Touch one side of the pipet with the heated glass bead. When the pipet tip fuses with the bead, slowly withdraw the pipet to pull a sharp bevel of about 5 |im in length (see Notes 11-15).

3.3.1.4. Making Transfer Pipets for 6.5-7.5 D Embryos

1. Heat the shaft of the Pasteur pipet over a flame.

2. When the glass begins to melt, take the pipet away from the flame, and hand-pull the pipet to produce a segment of thin capillary.

Fig. 5. (A) Schematic representation of the manipulation chamber used for manipulating 6.5-d embryos in hanging drops. The base of the manipulation chamber is made of glass of good optical quality supplied by the optometrist. The dimension of the glass strips supporting the coverslip is 3 x 3 x 30 mm. They are set between 4 and 6 mm from the edge. Two drops of solution are placed on the coverslip approx 1 cm apart, and then the coverslip is inverted over the paraffin oil-filled chamber. hp, Holding pipet; ip, injection pipet. (B) Schematic representation of the Petri dish set-up used for manipulating 7.5- and 8.5-d embryos. Two drops are placed approx 1 cm apart in the center of the dish and are then covered with paraffin oil. Angled pipets are used: hp, holding pipet; ip, injection pipet.

Fig. 5. (A) Schematic representation of the manipulation chamber used for manipulating 6.5-d embryos in hanging drops. The base of the manipulation chamber is made of glass of good optical quality supplied by the optometrist. The dimension of the glass strips supporting the coverslip is 3 x 3 x 30 mm. They are set between 4 and 6 mm from the edge. Two drops of solution are placed on the coverslip approx 1 cm apart, and then the coverslip is inverted over the paraffin oil-filled chamber. hp, Holding pipet; ip, injection pipet. (B) Schematic representation of the Petri dish set-up used for manipulating 7.5- and 8.5-d embryos. Two drops are placed approx 1 cm apart in the center of the dish and are then covered with paraffin oil. Angled pipets are used: hp, holding pipet; ip, injection pipet.

3. Break the capillary by bending the pipet. Check the size of the pipet tip under the dissecting miscroscope (0.5 mm for 6.5-d embryos and 1-1.5 mm for 7.5-d embryos).

4. Connected the pipet with a flexible plastic tubing to a mouthpiece. The movement of fluid and embryo in the pipet is controlled by suction or blowing into the mouthpiece.

3.3.2. Labeling the Epiblast Cells of 6.5-D Embryos with Carbocyanine Dye

1. Embryos are manipulated in a hanging drop under a Laborlux S microscope. Set up the micromanipulation chamber as in Fig. 5A.

2. Place a 5-pL drop of media (10% FCS in PB1) for holding the embryos during manipulation on a coverslip using a transfer pipet. A second 5-pL drop of dye is placed approx 1 cm from the first drop (see Note 11; Fig. 5A).

3. Invert the coverslip, and place it across the two glass strips of the chamber.

4. Fill the chamber with light paraffin oil.

5. To transfer embryos into the manipulation chamber, place the chamber under a dissecting microscope with the drops in focus. Pick up the embryos using a mouth-controlled transfer pipet. Pass the pipet through the oil to reach the drop, and gently expel the embryo with a small volume of medium.

6. Set up the manipulation apparatus as shown in Fig. 6. Attach the holding pipet to

Fig. 6. The micromanipulation assembly. The manipulators are clamped to the base plate. Each manipulator can hold up to two instrument holders. Movement of the instruments is controlled by screw-knobs (a,b) that allow positioning of the manipulator in the X-Y horizontal plane. There is a third control (c) by which the angle of inclination of the manipulator can be adjusted. Coarse and fine adjustment of the position of the manipulator in the Z-vertical plane is controlled by the fourth knob (d). Fine X-Y movement of the instrument under the microscope is controlled by joysticks (arrowhead). The holding instrument is on the lefthand side of the assembly and the suction and expulsion of fluid are controlled by the micrometer syringe (ms) on the right hand-side. Conversely, the manipulating (labeling or grafting) instruments are on the right-hand side and are controlled by the de Fonbrune syringe (fs) on the left-hand side. This set-up enables the positioning of the instruments and the control of syringe to be accomplished simultaneously.

Fig. 6. The micromanipulation assembly. The manipulators are clamped to the base plate. Each manipulator can hold up to two instrument holders. Movement of the instruments is controlled by screw-knobs (a,b) that allow positioning of the manipulator in the X-Y horizontal plane. There is a third control (c) by which the angle of inclination of the manipulator can be adjusted. Coarse and fine adjustment of the position of the manipulator in the Z-vertical plane is controlled by the fourth knob (d). Fine X-Y movement of the instrument under the microscope is controlled by joysticks (arrowhead). The holding instrument is on the lefthand side of the assembly and the suction and expulsion of fluid are controlled by the micrometer syringe (ms) on the right hand-side. Conversely, the manipulating (labeling or grafting) instruments are on the right-hand side and are controlled by the de Fonbrune syringe (fs) on the left-hand side. This set-up enables the positioning of the instruments and the control of syringe to be accomplished simultaneously.

the instrument holder on the left manipulator. Attach injection pipet to the instrument holder on the right manipulator. The holding pipet is controlled by the micrometer syringe on the right, whereas the injection pipet is controlled by the de Fonbrune syringe on the left. With this configuration, the positioning of the manipulator and the control of the syringe can be performed simultaneously.

7. Back-fill the holding and injection pipets with heavy paraffin oil by adjusting the syringes.

8. Position the manipulation chamber, and adjust the microscope to focus on the medium drop containing the embryos.

9. Bring the holding pipet into the field of view by pushing it through the oil into the drop. Draw a small amount of medium into the holding pipet to create an oil-medium meniscus.

Fig. 7. Labeling of the epiblast by microinjecting DiI into the distal cap of the embryo. To inject in the midline, the embryo is held at the distal tip of the egg cylinder by gentle suction with the holding pipet (h). The injection pipet (ip) passed through the extraembryonic tissues of the egg cylinder. The injection pipet is brought to the site of labeling from within the pro-amniotic cavity to avoid inadvertent labeling of other embryonic germ layers. The arrow points to the tip of the labeling pipet in the epiblast layer. en, primitive endoderm; *primitive streak. Bar = 20 |im.

Fig. 7. Labeling of the epiblast by microinjecting DiI into the distal cap of the embryo. To inject in the midline, the embryo is held at the distal tip of the egg cylinder by gentle suction with the holding pipet (h). The injection pipet (ip) passed through the extraembryonic tissues of the egg cylinder. The injection pipet is brought to the site of labeling from within the pro-amniotic cavity to avoid inadvertent labeling of other embryonic germ layers. The arrow points to the tip of the labeling pipet in the epiblast layer. en, primitive endoderm; *primitive streak. Bar = 20 |im.

10. Bring the injection pipet into view. Make sure that no medium is taken into the pipet, because the contact with an aqueous solution causes precipitation of the carbocyanine dye.

11. Retract the holding and injection pipets from the medium drop (Note 12). Bring the dye drop into the field of view by moving the stage of the microscope (Note 13).

12. Dip the tip of the injection pipet into the dye drop, and draw a small amount of dye into the pipet using the de Fonbrune syringe. Always keep the dye-oil meniscus in view, since this offers the only means for monitoring the flow of dye during labeling. Take the injection pipet out of the dye when the dye-oil meniscus has stopped moving.

13. Bring the medium drop containing the embryos back into the field of view.

14. Position the embryo by pushing it with the pipets and rotating it by lifting the pipet from beneath the embryo.

15. After the embryo is oriented correctly, bring the embryo into focus using the focusing control of the microscope, and then bring the injection and holding pipets into focus using the control knob on the manipulators (Fig. 6). Touch the injection and holding pipets lightly against the embryo to confirm that they are in the same plane.

16. Bring the holding pipet into contact with the endoderm of the embryo next to the epiblast to be labeled. Apply a suction force using the micrometer syringe to draw the embryo against the holding pipet. Increase the suction so that a small area of the endoderm layer is partly drawn into the holding pipet (Fig. 7).

17. Push the injection pipet through the extraembryonic tissue into the amniotic cavity (see Note 14). Then push the pipet tip into the epiblast layer (Fig. 7).

18. Apply pressure via the de Fonbrune syringe to expel a small volume of dye into the epiblast. It is important to monitor the movement of the dye front to avoid injecting any oil into the embryo.

19. Once the dye front has stopped advancing, retract the injection pipet (Note 15).

20. Release the embryo from the holding pipet by applying a positive pressure via the micrometer syringe. Return the embryo to the culture medium (Subheading 1.).

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