Determination of O2− production by H&E staining using confocal fluorescence microscope. Determination of O2− production by H&E staining using confocal fluorescence microscope. Cells were plated onto a glass slip in a two-well plate for 24 h, followed by pretreatment with candesartan (10 μmol/L), catalase (1,000 units/mL), or LY294002 (40 μmol/L). Cells were then treated with angiotensin II (10 μmol/L) for 5 h. H&E was applied to the cells 30 min before treatment was completed. After being stained, the cells were washed twice with PBS and fixed with 10% buffered formalin. The images were captured with a confocal fluorescence microscope. A. Control. B. Angiotensin II. C. Angiotensin II and candesartan. D. Angiotensin II and catalase. E. Angiotensin II and LY294002. F. Semiquantitative digital image analysis of O2− production by H&E staining in each panel. Hiroji Uemura et al. Mol Cancer Res 2008;6:250-258 ©2008 by American Association for Cancer Research