Figure 1: The formation of 8-nitroguanine (red) and the expression of Oct3/4 (green) were assessed by double immunofluorescence staining [10]. In the merged image, co-localization of 8-nitroguanine and Oct3/4 is indicated in yellow. Original magnification in all pictures is 200x (SH: Schistosoma haematobium). Formalin-fixed and paraffin-embedded biopsy and surgical specimens were obtained from normal subjects and patients with SH-induced cystitis and bladder cancer. Normal tissues and urinary bladder cancer tissues without SH infection were obtained from a commercial urinary bladder tissue array (Biomax.us, USA). Normal tissues with cystitis were excluded. SH-egg antigens in sera were detected by Sandwich ELISA assay [75]. This study was performed in accordance with the Ethical Guidelines for Epidemiological Research enacted by the Japanese government. Deparaffinized and antigen-retrieved sections were incubated first with 5% skim milk, and then with a rabbit polyclonal anti-8-nitroguanine antibody (2 μg/mL, prepared as described previously [11]) and mouse monoclonal anti-Oct3/4 antibody (2 μg/mL, Santa Cruz Biotechnology, CA, USA) overnight at room temperature. The sections were then incubated for 3 h with Alexa 594-labeled goat antibody against rabbit IgG and Alexa 488-labeled goat antibody against mouse IgG (each 1 : 400, Molecular Probes, Eugene, OR, USA).