Quantitative co-localization in immunostained cells. HeLa cells were immunostained with primary antibodies against the mitochondrial protein HSP60 and the TGN protein TGN46, followed by fluorescently-labeled secondary antibodies as indicated. (A) Primary anti-HSP60 antibodies were detected with a cocktail of two secondary antibodies, labeled with either Alexa488 or Alexa568. Co-localization analyses were carried out and the results are shown. The inset shows a four-fold magnification of the marked area, with arrows indicating membranes displaying more intense red labeling, and arrowheads indicating membranes displaying more intense green labeling. The RWC algorithm was able to detect these subtle differences with a higher sensitivity. (B) Cells were double immunostained with primary anti-HSP60 and anti-TGN46 antibodies and detected with secondary antibodies labeled with Alexa488 and Alexa568 respectively. Co-localization analyses were carried out and the results are shown. The inset shows a four-fold magnification of the marked area. Bars represent 10 μm. (C) Objects detected at 5 pixel and 500 pixel minimum sizes from the combined HSP60 and TGN46 images shown in panel B. Obj5px and Obj500px represent the mean co-localization coefficients (defined from the number of co-localizing objects as a proportion of the total number of objects) from the test set of images analyzed, at minimum object sizes of 5 pixels and 500 pixels respectively. The threshold of object size used has a significant effect on the co-localization coefficient determined.