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Counting NeuN cells

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Counting NeuN cells

cajal
Hi everyone!
I've been trying to count bunch of my hippocampal cells using NeuN staining. My first manual results were rather suspicious, which I think is due to the fact that I've probably counted background cells from lower layers.
I tried to do some corrections using a 8-bit inverted picture and then auto adjusted b & c. In a method I found, the authors then set the threshold to MaxEntropy. However when I do this in some of my sample no more cells are shown, only random single dots, which just can not be true!
Does anyone have a suggestion on how I can reliably count NeuN cells and be sure that I am not not counting the lower layers somehow? The problem is my sections are rather thick 40µm.
Best,
MJ