Stage VII of tail regeneration. Eublepharis macularius. (A,B) Gross morphology of the regenerate tail in lateral (A) and dorsal view (B). The regenerate tail is tapering cone that is longer than wide (length: diameter greater than 1.0). Serial sections stained (dorsal toward the top) with Masson's trichrome (C), immunostained with PCNA (D,E) or immunostained with Sox9 (F,G). (C) Transverse section through the regenerate tail. Similar to the original tail (see figure 2C), the regenerate appendage is concentrically organized with a central nervous system (ependymal tube, stained pink) enclosed by a regenerated skeleton (cartilaginous cone, stained green), surrounded by regenerated adipose tissue (unstained), regenerated skeletal muscle (stained red) and regenerated integument (dermis, stained green; epidermis, stained red). (E,F) Closer view of the interface between the cartilaginous cone and surrounding perichondrium immunostained for PCNA (brown), indicating proliferating cells, and counterstained with hematoxylin (blue). As the tissues differentiate and mature cell proliferation is reduced. In proximal portions of the tail (D), there are fewer PCNA-positive cells compared to more distal positions (E). (F,G) Closer view of the cartilaginous cone immunostained for Sox9 (brown), identifying differentiating chondrocytes, and counterstained with hematoxylin (blue). Chondrocytes in the proximal portion of cartilaginous cone (F) are fully differentiated and no longer express Sox9. Chondrocytes in the distal portion of cartilaginous cone (G) are positive for Sox9, indicating they are still undergoing the early phases of differentiation. cc, cartilage cone; pc, perichondrium; ra, regenerated adipose tissue; rd, regenerated dermis; rm, regenerate muscle. Scale bars: c = 500 μm; d = 200 μm; e-h = 20 μm.