Amputation of Limbs

1. Anesthetize the animals by placing them in a solution of 3-aminobenzoic acid ethyl ester (MS222) (Sigma). A concentration of 1 in 10,000 is required for young larvae, but older animals need a stronger solution, 1 in 1000. The acidic solution should be returned to neutrality with NaOH. Five to 10 min in this solution are usually enough.

2. Remove the animals from the anesthetic, place them on a wet paper towel under a dissecting microscope, and amputate the limbs at the desired level with a scalpel, razor blade, or scissors for larger animals.

3. Limb bones will protrude almost immediately owing to retraction of the skin and muscles from the wound surface. Trim these protruding bones, since they interfere with wound healing.

4. Return the animals to their tanks. It is better to keep experimental animals in individual bowls, since this prevents cannibalism, and they can be individually identified.

Fig. 1. Stages in the regeneration of adult newt limbs following amputation through the midlower arm (left side) or the midupper arm (right side). At the top are the original limbs. Stage 1—7 d; stage 2—21 d, stage 3—25 d, stage 4—28 d, stage 5—32 d, stage 6—42 d; stage 7—70 d after amputation. Redrawn from ref. 8.

Fig. 1. Stages in the regeneration of adult newt limbs following amputation through the midlower arm (left side) or the midupper arm (right side). At the top are the original limbs. Stage 1—7 d; stage 2—21 d, stage 3—25 d, stage 4—28 d, stage 5—32 d, stage 6—42 d; stage 7—70 d after amputation. Redrawn from ref. 8.

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