Welcome to the Forum! Let’s see if we can help you out…
So to start - it seems your main questions are not yet about colocalization analysis, but first properly segmenting your nuclei. So - for now you need to focus on Segmentation. There is a helpful “Segmentation in Fiji” workshop and corresponding slides that can help get you started.
Of course - you are going one extra step and wanting to only select those nuclei that have both DAPI and NeuN stains… so you will inevitably need to do a bit of scripting. Here are some other helpful links to get you started for that:
Once you have those regions selected - you can do colocalization analysis, using Coloc 2.
If I were you - I would start with this workflow:
- segment nuclei using DAPI staining - create ROIs for all DAPI+ nuclei
- move from ROI-to-ROI and measure NeuN staining - set whatever threshold you want and based on that, save or delete that ROI
- save those ROIs and then use them to apply within Coloc 2 for your subsequent analysis.
Perhaps someone else has a better way to proceed… but this will at least get you started. If you have more specific questions - just let us know.