Dissection of Apical Ridge and Grafting to Limb Bud Fig 3A

4. Dissect apical ridge of the limb bud away from underlying mesenchyme using fine needles.

5. Place apical ridge with small amount of adherent mesenchyme into ice-cold trypsin (see separation section) for 5-10 min. This should be sufficent time to loosen tissues.

6. Transfer tissue to ice-cold tissue-culture medium containing serum and peel apical ridge away from mesenchyme in ice-cold tissue-culture medium.

7. To graft apical ridge to the dorsal surface of a wing bud, cut a shallow slit running from anterior to posterior in the dorsal surface near the tip of right wing bud (good stages are 20-21) with a sharp needle.

treat with trypsin to separate mesenchyme and ectoderm treat with trypsin to separate mesenchyme and ectoderm

Fig. 3. (continued) (B) Diagrams to illustrate how mesenchymal and ectodermal components of limb buds are separated, recombined and grafted to allow further development (Subheadings 3.1. and 3.3.). Separate ectoderm and mesenchyme of chick limb buds by soaking in 2% trypsin for 1 h at 4°C and then placing in tissue culture containing serum. Recombine mesenchymal core (M) from one limb bud with ectodermal hull (E) from another bud. Allow tissues to reanneal at 37°C for 1 h, and then graft recombined tissues either to wing bud stump and pin in place or to dorsal surface of older wing bud.

Fig. 3. (continued) (B) Diagrams to illustrate how mesenchymal and ectodermal components of limb buds are separated, recombined and grafted to allow further development (Subheadings 3.1. and 3.3.). Separate ectoderm and mesenchyme of chick limb buds by soaking in 2% trypsin for 1 h at 4°C and then placing in tissue culture containing serum. Recombine mesenchymal core (M) from one limb bud with ectodermal hull (E) from another bud. Allow tissues to reanneal at 37°C for 1 h, and then graft recombined tissues either to wing bud stump and pin in place or to dorsal surface of older wing bud.

8. Transfer isolated ridge into host egg, maneuver over slit, and poke into slit with a blunt needle.

Fig. 3. (continued) (C) (Subheading 3.4.) Diagram to show how a polarizing region is grafted beneath the apical ridge at the anterior margin of a chick limb bud. Cube of posterior mesenchyme cut from donor limb bud. Site for graft prepared in host limb bud by cutting along base of apical ridge, starting at arrow, and then easing ectoderm away from mesenchyme. Cube of posterior mesenchyme placed beneath anterior loop of apical ridge.

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