After the stated period of time, the animals were reanaesthetized and the regenerating limbs were amputated at the shoulder, stained, mounted and photographed. Within 24 hours of amputation, a layer of epidermis migrates across the limb stump, closing the wound. This is a critical step for limb regeneration, since close contact is necessary between the epidermis and underlying muscle tissues in order for regeneration to occur. After the wound is closed, the cells of the stump undergo a process of dedifferentiation. For example, muscle cells lose their contractile apparatus and return to the mononucleate condition.
Dedifferentiation occurs for some distance proximal to the amputation level.
The dedifferentiated cells then begin to accumulate at the end of the stump beneath the epidermis to form the blastema. In the blastema, mitosis occurs rapidly and the combination of migration of dedifferentiated cells and the proliferation of these cells results in a large mass of morphologically undifferentiated cells. This mass of cells then begins to elongate to form a cone. Later, the cone flattens to form a paddle or palette-shaped regenerate. Once the cone begins to elongate, one can usually identify cells that are beginning to redifferentiate.