Quantitative co-localization analysis of transport through the early secretory pathway. HeLa cells were transfected with plasmids encoding the secretory cargo CFP-ts045G. Following incubation at the restrictive temperature for ts045G release from the endoplasmic reticulum, the temperature was reduced to allow folding and release of the cargo into the secretory pathway. Cells were fixed at various time points, immunostained for markers of the cis-Golgi complex (GM130) or the TGN (p230), and co-localization analyzed using the RWC algorithm. (A) Example images from various time points of release showing CFP-ts045G (green) moving from the endoplasmic reticulum (5 mins), through the Golgi complex (20 and 30 mins), and beginning to arrive at the cell surface (60 mins). Bar represents 10 μm. (B) RWC co-localization analysis of ts045G with GM130 and p230 demonstrated an initial peak of the cargo with the cis-Golgi marker at 20 mins, followed by a peak in co-localization with the TGN marker at 30 mins. The RWC algorithm was sufficiently sensitive to record this subtle change in localization. Error bars indicate mean and standard deviations between the 10 cells analyzed for each time point.