Hippocampal neuron quantification

Hello everyone,
I am trying to quantify the number of neurons in different subregions of the hippocampus which is stained by NeuN. I encountered two problems: 1. the contrast is not consistent in my image so the CellProfiler is picking up some background stuffs that are not neurons (like below). 2. I want to count how many neurons in a confined area, not the whole image, how can I draw that out? I tried using ‘IdentifyObjectsManually’ along with ‘IdentifyPrimaryObjects’ but it is not working. Thank you!
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I am attaching some of my original images:


The pipelines I am using: NeuN IHC trial 8.12.2019.cppipe (22.4 KB)

Hi, Open your image in ImageJ and outline the region you want:

convert that outline to a “mask”:

save it:

then, in the CP pipeline import it along with the neun label image:

use the MaskImage module to “mask” the image with the NeuN label:

now do the segmentation for NeuN objects on the masked image:

and CP will confine the segmentation to the region specified in the mask.