Getting started, UnmixColors, SaveImages, Identify objects

I am entirely new to image analysis. I am studying virus attachment protein/receptor interaction. I have some mutant viral attachment proteins, and want to compare binding of mutant and wt attachment proteins to tissues. The bound attachment protein is identified by AEC staining. I ultimately want to quantitate (and compare) the intensity of AEC staining. The tissues are counterstained with hemotoxylin. So I think my first step is to Unmix colors into AEC and hemotoxylin. This seems to be working for hemotoxylin, but I am unable to get the AEC image to save. This is true even if I write a pipeline with just AEC and no hematoxylin.
I have attached the pipeline I have created so far and some sample images I will be evaluating and would greatly appreciate some help.
My next problem will be how to identify stained “objects” so I can compare intensity.
I am not interested in the number of cells stained. It is probably impossible to distinguish between individual cells stained in the tracheal epithelium.
I am not interested in measuring the area stained (although to determine mean intensity of stained areas, this would be necessary).
Different tissues have different staining patterns, so I will likely need different pipelines for different tissues.
The “objects” I want to identify will vary widely in size and shape. (In tissues where more than one kind of cell is staining, it might be of interest to evaluate intensity of staining of the different kinds of cells separately.)
There is also the issue of background staining, both of the slide where there is no tissue and of some other surfaces of the trachea tissue other than the ciliated epithelium. I need to be able to subtract the background.
test unmix colors.cpproj (403 KB)











I have figured out in the meantime why the images would not save. The step in the pipeline was not checked. I have also figured out how to select objects. I just have to set the diameter low enough to pick up everything I see stained. I have been exploring the threshold intensity and minimum object size I need to use to identify the stained areas and seem to be having success.

Great, thanks for writing back. Let us know of you have further questions.
Best,
David