Grafting Paraxial Mesoderm and Somites In Ovo 21 Materials

1. Dissecting kit: 2 pairs small forceps (watchmaker's; number 4 or 5), 1 pair small scissors (about 2 cm straight blades), 1 scalpel (No. 3 handle, No. 11 blade), a Gilson micropipet for 3 ||L, yellow tip(s).

2. Eye surgeon's micro-knife, 15° angle. Suitable microknives are: Microfeather microsurgery scalpels for eye surgery, 15° blade angle, cat. no. 715, manufactured by Feather (Japan) and marketed by pfm GmbH. Sold in boxes of 5, not cheap!

3. 2 Entomological pins, size A1 or D1, or sharpened tungsten needles, each mounted by melting the end of a short Pasteur pipet (to act as handle) or inserted into a metal needle holder.

4. Plasticine (modeling clay) to make ring for resting egg on its side.

5. 100 mL Calcium-/magnesium-free Tyrode's saline (CMF): dissolve: 80 g NaCl, 2 g KCl, 0.5 g NaH2PO42H2O, and 10 g glucose in 1 L H2O. Autoclave for storage. On day of use, dilute 1:10 with distilled water. The working solution may be buffered with bicarbonate, but we usually omit this.

6. Antibiotic/antimycotic solution, 100X concentrate (e.g., Sigma A9909; St. Louis, MO).

7. 70% Ethanol to wipe shell.

8. PVC tape to seal egg.

9. 2 Pasteur pipets and rubber teats.

10. Container for egg waste.

11. 10-mL Plastic syringe with Gilson yellow tip stuck into the end, filled with high vacuum silicon grease or vaseline.

12. 1-mL Syringe, 27-gage (or finer) x 3/4 in. needle (for ink injection).

13. 1-mL Syringe, 21-gage needle (for antibiotics).

14. 5-mL Syringe, 21-gage needle (for withdrawing albumen).

15. Paper tissues.

16. 35-mm Plastic dish coated with Sylgard and steel insect pins, size A1 or D1.

Sylgard 184 (Dow Corning) is clear silicone rubber polymerized by mixing two components (9 parts rubber solution:1 part of accelerator/catalyst). Mix the two well and pour to the desired depth (2-5 mm) into the plastic Petri dish. Allow the dishes to stand for about 1 h at room temperature for air bubbles to leave, then cure at about 55°C until polymerized (3 h to overnight). The dishes can be stored indefinitely. Black Sylgard is also available.

17. India ink (Pelikan Fount India is best; most other makes are toxic), diluted 1:10 with CMF and loaded in the ink injection syringe.

18. 50 mL Trypsin (DIFCO, 1:250), freshly made up to 0.12% (w/v) in CMF.

19. Hens' and quails' eggs incubated 40-44 h so that they are at stages 10-12. The hens' eggs (hosts) should have been resting on their sides at least 30 min.

20. Dissecting microscope, preferrably with transmitted light base.

21. Fiber optic incident illumination.

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