I just don't know what to do with ... values "0"

I have a problem with Fiji results, how can I adjust “detection” in this case, any piece of advice? Any method while optimization process using IHC plugin maybe?

Some of my results are like this:
Total count: 1896
Positive count: 0
Percent count: 0
Total area:4946.6509
Positive area:0
Percent area: 0
Average intensity: 0

But with looking at the pictures the staining is not zero… Which parameters should I change?

Could anyone here help me? I know it’s pandemic out there (for me personally it’s hard to focus) - still would be thankful for the advice

Hi @Katarzyna_Kaleta,

can you share an insight to your data and/or the workflow you are performing? Otherwise it’s super hard to come up with meaningful suggestions :wink:


Hi, I’m counting positively stained cells in human skin. There should be differences between 2 groups with lower expression of PPR-gamma in obese group. I have 2 IHC DAB stained photos for each patients… I can show sample pic / or excel with calibrations results with Andy… but this forum does not allow to attach xlsx format apparently - on e-mail maybe? If you could be so nice to help me?

With “workflow” I mean: What are you doing in Fiji? And how do your images look like? Without knowing that, we cannot tell you what to do differently :wink:

Hi @Katarzyna_Kaleta,

Is this post related to what you are trying to achieve?

Ideas on Quantifying Positive Nuclei?



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Ok, but I’m not sure If I’m doing it correctly.
I attach 5 sample photos, of 2 different receptors DAB staining.
I’m using fiji Andy’s algorithm plugin - IHC.
First I analyzed photos for calibration for each receptor separately. Some stainings are weaker, some are pretty obvious. Next, I put parameters suggested by the calibration process and analyzed a whole group of photos. As I said some results are “0” but for me staining is positive…? How can I obtain better detection? And make the results more reliable? I see staining in nuclei, in the cytoplasm - does it influence the method? I want to detect all.
What particular information should I give so that we can solve the puzzle?

Plus, an additional question on statistics: let’s say that staining is “0” how should I put it into statistics? The lower value of average intensity says that the staining is stronger. So 0 average intensity will make a mean value paradoxically more, when there is less…

I would be extremely thankful for help!

18.02,pprgamma1-100,ph9,489-17,7_TRANS_0001.tif (4.0 MB)
21.02.20,igf1r685-14,20x4_TRANS_0001.tif (4.0 MB)
21.02.20,igf1r1696-15,20x4_TRANS_0001.tif (4.0 MB)
21.02.20,igf1r186-18,1-10,9999,20x,5_TRANS_0001.tif (4.0 MB)
18.02,pprgamma1-100,803-19,ph9,6_TRANS_0001.tif (4.0 MB)

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