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foreign open the image of Interest using image J software the first step is to split the channels of the image at this point the green channel can be disregarded select the blue Channel dappy and adjust the threshold until all the nuclei are red in color with a black background once adjusted select apply to separate the nuclei from one another click on process binary and watershed now the nuclei are separated from one another select all the nuclei by clicking on edit selection and create selection save this image as selection rename the file and save it with a DOT Roi extension now this window can be closed select the red Channel and duplicate the image since we require two images one for analyzing the nuclear intensity for instance the right hand side image and the other for cytoplasmic staining intensity for instance the left hand side image adjust the threshold in both of these images such that the foreground is completely red in color with a dark background once the threshold is se
