Double immunolabeling for dopamine transporter (DAT) (green) and tyrosine hydroxylase (TH) (red) in Subjects 1 (panels A–E, graft survival of 4 years), 2 (panels F–J, graft survival of 4 years), 4 (panels K–O, graft survival of 9 years), and 5 (panels P–T, graft survival of 14 years). Panels A, F, K, and P show a low magnification composite for each subject to illustrate the grafted cell bodies and adjacent reinnervated putamen. Successively higher magnification images are illustrated in panels B–D, G–I, L–N and Q–S. Boxed areas represent the image shown in the subsequent panel. Panels E, J, O and T show a low magnification composite of the patient’s putamen and adjacent external segment of the globus pallidus. DAT immunostaining shows a robust punctate localization along dopaminergic (TH-immunoreactive) fibers in the reinnervated putamen and caudate in all grafts, even up to 14 years post-transplantation. In addition, the transplanted dopamine neurons show a healthy and non-atrophied morphology. Parallel control immunostainings, where the primary antibodies were omitted, showed no immunoreactivity of DAT or TH (data not shown). To further confirm specificity of the DAT labeling observed in the reinnervated putamen and caudate, we also examined DAT immunolabeling in adjacent anatomical regions on the same tissue sections. In the lateral and medial globus pallidi, which are regions that receive comparatively little dopaminergic innervation and normally exhibit little DAT expression in the human brain (Ciliax et al., 1999), we, as expected, observed weak DAT immunolabeling and a sharp boundary from high to low DAT and TH expression (panels E, J, O, T). We also performed immunostaining in the substantia nigra of each patient and confirmed the loss of both DAT and TH immunoreactivity (data not shown). g = graft, h = host, GPe = globus pallidus externa. Scale bars in A, E, F, J, K, O, P, T = 400μm; B, G, L, Q = 100μm; C, H, M, R = 50μm; D, I, N, S = 20μm. See also Figure S1.