I am looking for a software (&the protocols) that I can use to count the area of positive staining (the brown one, in ECM) & total area of tissue (in fibrous and bone tissue). I have tried ImageJ (the result seem not valid because the positive staining is bigger than the total) and now trying QuPath (but I don't know the protocols). So can anyone recommend another software? or know how to use those two softwares properly? or have a traditional method (like use intensity). Thank you before!
Is your goal to measure the percentage of total area that is comprised on the ECM?
My suggestion is to work more with ImageJ or Fiji (Fiji is just ImageJ with plugins pre-loaded). There are many tutorials for ImageJ-the one I uploaded is only one example.
Make sure that you threshold the image properly. And also make sure that the threshold was applied before measurement so that the results are set to threshold.
@Khalid Eldahan, thank you for the answer but I have tried ImageJ but it didn't work because I use only light microscope and the picture doesn't show nucleus that good.
It's impossible to provide advice without atleast a sample image. ImageJ/FiJi more than likely can handle the job, assuming the source images are of sufficient quality.
@Peter Sobolewski This is a M. nemestria section that I processed with IHC. I want to count the area of positive staining (the one that brown-ish) over the total area.
I think you should be able to get what you want using Color Threshold in ImageJ/FiJi (Image menu, Adjust > Color Threshold).
You can stick with HSB color mode (the default)
To select the brown, just gate the top on the greenish and purplish peaks, then uncheck the box Pass to reject those pixels (eg. 47-220).
You can leave Saturation more or less wide open, then gate down the Brightness to eliminate pure black on the left and the white on the right. Adjust till you get a decent selection. You can click Original and Filtered to see what it looks like and use the zoom to look closer at tricky regions.
Once you have something click Select and then menu Analyze > Measure.
To clear the selection just go to Edit > Selection > Select None.
To get total, you just need to reject the background, the green, so just reduce your first gate in Hue to cover just the green peak (eg. 47-133) and widen your Brightness until you get the whole tissue.
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