Detection of RASSF1A protein in clear cell RCC and control cell lines. Paraffin-embedded tissue samples obtained from renal tumor and paired normal tissues were analyzed for presence of RASSF1A protein using immunohistochemistry (counterstaining hematoxylin). a. Immunohistochemical positive control: CaSki cell line demonstrating absence of RASSF1A promoter methylation in COBRA (see box; U, unmethylated signals; M, methylation signals) and immunopositivity depending on the absence (i) or presence (ii) of an antibody blocking peptide, respectively. b. Immunohistochemical negative control: HEK293 cell line showing strong methylation in COBRA (see box; U, unmethylated signals; M, methylation signals). Only residual unspecific nuclear immunopositivity can be observed, which is independent from the absence (i) and presence (ii) of an antibody blocking peptide. c. Tumor cells of a clear cell RCC (magnification, 400×). d. Histopathologically normal tissue with immunopositive epithelial tubular cells (magnification, 400×). e. Heterogeneous staining patterns demonstrating immunopositive (i) and -negative (ii) epithelia in renal tubules. f. Mosaic-like staining signals in a single tubular epithelium (see arrows, magnified from panel d as indicated).