Nuclear staining analysis

Hello,
i’d like to do analysis of the nuclear staining of phosphorylated NF kB, so that i can prove the difference between two samples the control and the treatment.(if the treatment increase the phosphorylated NF kB in the neuclei or not). I tried this methode (CTCF = Integrated Density – (Area of selected cell X Mean fluorescence of background readings)), but unfortunately it gives me negative numbers.
this is an example for the pic, that i’ve to analyse:

Thank you

Hi Alen and welcome to the forum :slight_smile:

Look into colocalization analysis and specifically the Coloc 2 plugin. You can perform this colocalization analysis on your nuclei specifically by segmenting the DAPI stain image and creating ROIs that define your nuclei.

I am sure @etadobson (developer of Coloc2) will be happy to help with the colocalization analysis if you need it.

PS. in general it is helpful if you could share a raw data example so that users of the forum can test various approaches to solve your problem directly.

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thank you for your help.
Actually what i want to do is like this study:

The link of study: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225449/
But i couldn’t find a protocol how to do it and i don’t have that experience in ImageJ.

So, this figuretext outlines the approach they used to segment their images, you could try to follow the same procedure, but consider that it may not be ideally suited to your data. If you can share some raw images, users of the forum will be better able to suggest which segmentation methods might suit your specific needs.

But, I am sure you could figure out a lot of it on your own, there are many resources available on the ImageJ wiki:

And of course, please post your questions if you get stuck :slight_smile:

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