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Fig. 1. Expression of Wt1 mRNA and protein in human retinoblastoma. Tissue samples were obtained from six patients diagnosed with moderate to poorly differentiated retinoblastoma (RB1 to RB6) according to the classification of Nork et al. (23) . RT-PCR was performed with 1 µg of total RNA each. The number of PCR cycles was adjusted to 28 to be within the exponential phase of the semiquantitative PCR. Note that Wt1 transcripts were detected in all tumor samples examined (A). B–E, representative Wt1 immunohistochemistry of human retinoblastoma. Wt1 protein was detected using a second antibody conjugated with alkaline phosphatase as a substrate (purple staining). Nuclei were counterstained with hematoxylin (blue staining). The low power magnification (B) shows normal (right) and neoplastic retina (left). High power magnifications (C and D) revealed Wt1-positive cells in the more differentiated parts of retinoblastomas, especially in areas with Flexner-Wintersteiner rosettes (D). Note also that Wt1 immunoreactivity is restricted to tumor regions with low proliferative activity, as demonstrated by double-labeling with the proliferation marker Ki-67 (E), which is visualized by a brown color reagent. Wt1-positive cells (purple) were detected in regions with low Ki-67 immunoreactivity. For negative controls, the primary antibody was replaced by normal serum (F). The immunohistochemistry technique was validated by demonstration of nuclear Wt1 staining of glomerular podocytes in tissue sections from human kidney (G).