First, BEFORE POSTING - Please read this
Basically, a pipeline would be:
(1) LoadImages (DAPI and Ab)
(3) IdentifyPrimaryObjects (DAPI).
?? Is the Ab in nuclear compartment, as well as the cytoplasm?
(4) IdentifySecondaryObjects, using the nucleus as the seed, and the Ab channel to “grow” into cytoplasm (try “Propagate”)
(5) MeasureObjectIntensity - measure Ab in either nuclear and/or cytoplasm
We have example pipelines here: cellprofiler.org/examples.shtml
These cells are fairly confluent, so don’t expect great segmentation. You can post your pipeline here and we can comment on it.