Materials

1. Roll of clear adhesive tape (Sellotape) and roll of thick plasticized canvas tape, both 25-mm wide and on a dispenser.

2. 27-gage x 21 mm, 19-gage x 35 mm hypodermic needles; 1- and 5-mL syringes.

3. Egg stools, cut from flexible plastic sleeve used for insulating water pipes.

4. 2 Sylgard-bottomed Petri dishes (1 for fixed material and 1 for live). Sylgard (Dow Corning, Wiesbaden, Germany) is a silicone resin that comes in two parts. When mixed, it sets to produce a near-transparent rubbery matrix, ideal for pinning down embryos. To prepare Sylgard-bottom dishes, premix Sylgard, pour into 35- or 60-mm plastic Petri dishes, and allow to set. Store the dishes at room temperature, sealed with Parafilm. Use with stainless-steel minuten pins.

5. Howard's Ringer. 0.12 M NaCl, 0.0015 M CaCl2, 0.005 M KCl/L: 7.2 g NaCl, 0.17 g CaCl2, 0.37 g KCl. Adjust to pH 7.2, with very dilute HCl and autoclave.

6. Penicillin/streptomycin/fungizone (PSF) (Gibco-BRL, Paisley, UK). Aliquot (500 |L) and store at -20°C.

7. Dispase (Boehringer, Lewes, UK). 1 mg/mL in tissue culture medium, such as F12 or L15; 500-|L aliquots. Store at -20°C.

8. DNase I (Boehringer, Lewes, UK) for enzymatic preparation of notochord according to Subheading 3.3.2., step 2: 1 mg/mL stocks in Howard's Ringer, aliquot (100 |L). Store at -20°C.

9. Sterile fetal calf serum for coating Gilson tips, 50-|L aliquots. Store at -20°C.

10. L15 tissue culture medium (for enzymatic preparation of notochord according to Subheading 3.3.2., step 2).

11. India ink (e.g., Pelikan "Fount"-lacks shellac, nontoxic).

12. #11 Scalpel blade and handle or commercial egg pricker.

13. Pair of curved, sharp-pointed scissors.

14. Pair of Vannas spring scissors.

15. Pair of ultrafine scissors.

16. Pair of coarse forceps.

17. 2 pairs of fine watchmakers' forceps (#5 or 55).

18. Stainless-steel spatula, 5- to 8-mm wide tip. Bend to obtain a 135° elbow.

19. Stainless steel minuten pins (for Sylgard dishes).

20. Pure tungsten wire, 100 |im.

21. Four needle holders (6 x 120 mm glass capillary rod).

22. Sealing wax.

24. 60-mm Bacterial Petri dishes.

25. Long glass Pasteur pipets.

26. Thin silicone tubing plus adapter.

27. Bunsen burner.

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