YFP-WT PINK1 (green) shows mitochondrial localization in HeLa cells following treatment with the mitochondrial depolarizing agent CCCP (carbonyl cyanide m-chlorophenyl hydrazone). When fixed cells are unpermeabilized (not treated with detergent), antibodies to GFP (red) (used to identify YFP-WT PINK1), and Tom20 (white), a mitochondrial outer membrane marker, are unable to reach and bind their epitopes. YFP-WT PINK1 transfected HeLa cells were treated with CCCP (10µM) for 3 hrs, fixed with 4% paraformaldehyde and not permeabilized. Primary antibodies used were: anti-GFP polyclonal Ab (Invitrogen) and anti-Tom20 mAb (BD); secondary antibodies used were: Alexa Fluor 594 and 647. Imaging was performed on an LSM510 Meta (Carl Zeiss, Inc) with a 63x 1.4 NA oil differential interference contrast Plan Apo objective. Image contrast and brightness were adjusted in the LSM image browser (Zeiss). This image corresponds to Fig 4c, top row, and is a control experiment for a differential permeabilization assay to determine submitochondrial localization of PINK1 and that is further described in Fig 4c of J Cell Biol, 191: 933-942, 2010. Images in Fig 4 include CIL#s 13733, 13734, 13729, 13730, 13731, 13732, 13717, 13718, 13719, 13720, 13721, 13722, 13723, 13724.