Getting Started

1. Incubate eggs. Generally, chick embryos between HH 8-13 can be used for notochord transplantations. To study floor plate induction, use HH 9-10. If you plan to use the mechanical method of notochord preparation (Subheading 3.3.1.), incubate some eggs until HH 13-16 as donors. Despite these guidelines, the ideal stages of hosts and donor has to be determined according to the specific aim of the study. Follow the guidelines for storage and incubation of eggs elsewhere in this volume (Chapter 14). Lay eggs on their sides when incubating for manipulations. Mark this side with a pencil.

2. Microscope: Set up a stereomiscroscope with fiber optics. Check that the light source has a heat filter. Hand-rests to steady the hands may be useful. Have a Bunsen burner within reach.

3. Thaw one aliquot each of PSF, Dispase solution, and FCS. If you are using method II to isolate the grafts, also thaw DNase I.

4. Make up 50 mL of Howard's Ringer with 1:100 PSF.

5. Prepare contrast medium: 1-5 mL of India ink, diluted 1:5 in Howard's RingerPSF. Draw into a 1-mL syringe, attach a 27-gage x 21 mm needle, expel any air, and bend the last 1 cm of the needle into a 60° angle.

6. Prepare operation needles. Warm up one end of the glass rod with the Bunsen burner, and then dribble some molten sealing onto the tip of the rod. Do not set fire to the wax. Cut 20-mm lengths of tungsten wire from the reel, and straighten out the curve in the wire. Hold the wire with coarse forceps, and then fasten the wire to the glass rod by pushing it through the soft wax into the hole within the rod. Bend 4 mm at the free end of the wire to form a 135° elbow. Holding this tip into the hottest region of the Bunsen flame (blue color) and keep it there until the last 1-2 mm flies off. Withdraw the needle from the flame immediately—it will have a very fine sharp tip. Be careful not to touch anything hard with your needle, since this will ruin the tip.

7. Prepare a thin glass capillary. Hold capillary of Pasteur pipet over the Bunsen flame, and bend an angle in the middle of it. Warm the portion between this angle and the main part of the pipet, and then pull to obtain a very thin capillary. Break the rest of the pipet off, leaving about 2 cm of the thin capillary behind. The coarse end of this capillary can be attached to the adapter of the silicone tubing to form a mouth pipet.

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