Just one last question; let's say your interest in just the area of DAB-staining without analysing the nuclei at all, would there be any effect to the amount of DAB quantify? Granted that you've found the appropriate threshold levels. I noticed that the number of DAB regions were not affected at all in my analysis, only the number of nuclei due to segmentation.
I don't totally follow the question- do you mean would the masking of empty areas affect the DAB segmentation? Again, it's impossible to predict ahead of time, the only way to know is to try it, it might make DAB segmentation better, worse, or have no effect. I'm not sure what that would get you though- you'd still have to go through the same process of identifying the area of the DAB foci, identifying the area of the empty areas, and doing the same calculations as if you hadn't masked at all.
If I've misunderstood the question, please let me know.