Separation of Ectoderm and Mesoderm Components of Limb Buds Fig 3A

This can be accomplished for limb buds over a range of developmental stages from stage 19 to 29 (29).

1. Remove chicken embryo from egg through window in shell. To do this, first cut a circle into the yolk through the vitelline and chorionic membranes around the embryo, and then lift out embryo on a small spatula.

2. Place embryo in Petri dish containing PBS or tissue-culture medium (that equilibrates with air), and pull away the membranes surrounding embryo with a fine pair of forceps.

treat with trypsin to separate apical ridge and mesenchyme poke apical ridge into slit

poke apical ridge into slit

Fig. 3. (A) (Subheading 3.2.) Diagrams to show how an apical ridge is isolated and grafted to the dorsal surface of a limb bud. Remove thin sliver of donor limb bud tip, and soak in 2% trypsin at 4°C for 5-10 min. Separate apical ectodermal ridge, and keep in ice-cold medium. Graft ridge to slit made in dorsal surface of host limb bud, and poke into place with blunt needle.

3. Remove limb buds from embryo using fine forceps to pinch through tissue where buds attach to body wall.

4. Prick ectoderm of limb buds with fine needles ,so that trypsin will penetrate easily to bud tip.

5. Place limb buds in ice-cold 2% trypsin, and leave on ice for between 30 min and 1 h (precise length of time will depend on size of limb bud and batch of trypsin; ref. 30).

6. Transfer limb buds to ice-cold tissue-culture medium containing serum and leave on ice for 5 min. (Serum contains trypsin inhibitor and will "stop" trypsin)

7. Place Petri dish containing limbs on a cold slab, and ease loosened ectoderm from mesenchyme by inserting a needle between the tissues and working from cut edge of ectoderm. It should be possible to remove the ectoderm, which looks like a diaphanous mitten, with attached basement membrane (31).

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