I am new to CellProfiler and got stuck with my analyzes.
Background: I have Myotubes on a cellular level which are expressing eGFP and are stained against alpha actinin and DAPI. I would like to compare the levels and localization of GFP expression to alpha-actinin. In my opinion DAPI won’t help that much, because the myotubes are multinucleated.
So far, I could manage to identify alpha-actinin as my primaryobject but I am stuck with my secondary object GFP. I am able to detect most GFP expression, nevertheless the strongest expression is not detected – somehow feels like it is above a certain threshold. I already played around with lots of settings. Still I was not able to detect the brightest GFP expression. All topics related to muscle-staining have a cross section and no longitudinal tubes.
Thanks in advance for your help, I will support my pictures in the second post.