Triple fluorescent staining analysis



Hi all!
I have a triple fluorescent staining (DAPI nuclei in blue, Ki67 nuclei staining in red and Mac3 staining membrane an citoplasm in green). I would like to quantify the amount of cells positive for Mac3 and Ki67. I managed to quantify cells positive for DAPI and Ki67 because those two staining colocalize, but I don’t know how to then screen for only cells Mac3 positive. Is there anyone who has an idea? I usually work with Fiji, but if Cellprofiler is a better option I can download it :slight_smile:
I upload some images that I hope will help with understanding the problem.

Thank you very much,


You could possibly split the image into color channels and only use the channel which shows the Mac3 cells the best. Meanwhile I will try to work on a better way.



Hi Bob,

thank you very much for reading my post and helping out. I realized I didn’t write really in a clear way, but what I want to quantify is the number of Mac3 positive cells that are also positive for Ki67.

Thank you very much.


Hello Laura,
You stated in your earlier note that you wanted to segment areas tested both Ki67 and Mac3 positive. Here is an inhanced image which shows the Ki67 and Mac3 positives which show as Yellow (combined R+G ). The procedure is fairly involved, but if it works for you I can explain further.



Hi Bob,
It looks really good! Thank you very much for spending time on it. I’d love to learn more about how it works.



O.K. Laura,
Here we go, select your image (I used the one you titled as Merged). Then goto Edit > Selection > Select All.
Goto Analyze > Plot profile (enter) and put your mouse over the lowest point on the plot, get the value of ‘Y’ from the title bar at top. ( the one in the plot window reads differently) remember this value.
Goto Process > Math > Subtract > the Y value you obtained previously.
Goto Analyze > Plot profile > and do the same thing as before except obtain the highest value.
Goto Process > Math > Divide > the Y value (high)
Goto Process >Math> Multiply*250
The above process will give you the best enhancement you can get (unless you do it multiple times). You should see the Mac3 and Ki67 (both positive) as yellow for R+G. Segment with Image> Adjust> Color Threshold with Blue @ 0, and both R and G set equally @ 150-200. Then Analyze> particles for the math.
This should have been easier, but the Process> Math> Macro does not seem to be functioning properly in my installation (unknown reason).

Hoped this helped,