I am a new user (and, therefore, cannot upload attachments). The goal is to use Cell Profiler to quantify the percentage of the total cells (total DAPI) that are TH positive. I have z-stacks with a TH channel and a DAPI channel. The basic Pipeline is: Threshold for TH to eliminate faint TH staining in the neurites, Identify Primary Objects for DAPI, Identify Primary Objects for TH, Relate Objects (TH Parent - DAPI Child), Classify Objects. I am experiencing a few issues: (1) I am having trouble accurately segmenting TH cell bodies. Thresholding eliminates the TH staining in the neurites but it also eliminates more faint TH cell bodies. I tried setting it up such that the TH cell bodies are Secondary Objects to the DAPI Primary Objects but that was not effective. (2) I am having trouble with the Relate and Classify Object Modules; specifically, they are not yielding the output that I would like; namely, count for TH-DAPI doubles and DAPI singles. Any tips or suggestions are appreciated.