IHC problems identifying negative cells

Hello, I am analyzing my IHC image stained with DAB and hematoxylin. I am using the wound healing example pipeline, but it is just identifying the positive cells and not the negatives, I tried changing the threshold but I end up selecting everything or none. I want to know the percentage of positive cells within th epopulation. The problem is that I have on my image also blank spaces where the bone was and I do not want to consider those areas. Can you help?

thanks

kena

HELLO, I JUST FOUND THE UNMIX COLORS MODULE AND IT RESOLVE ALMOST ALL MY PROBLEMS. IT IS ASSIGNING BLACK COLOR TO THE AREAS THAT HAVE NO CELLS OR WHERE THE BONE WAS, WHICH IS WHAT I WANTED. I STAINED THE CELLS WITH DAB AND HEMATOXYLIN, AND USING EITHER OF THOSE STAINS ON THE MODULE GIVE ME SIMILAR RESULTS, I FOUND THAT SOMETIME IT RECOGNIZED THE STRONG HEMATOXYLIN CELLS AS POSITIVE FOR DAB, DO YOU THINK IS BETTER TO NOT COUNTERSTAINED THE CELLS? CAN THE SOFTWARE STILL RECOGNIZED UNSTAINED CELLS?

THANK YOU :smile:

Hi Kena,

Color unmixing can be tricky because what we assign as a single color to our eyes actually represents a range of colors that the module is being asked to identify and distinguish. So I think counterstaining should be fine provided you are convinced that it’s working adequately. If you’re still working on your counterstaining protocol, a bit of googling turned up this link; I’m sure you could find more.

If the counterstaining is working well, then sometimes you can have better results by using the ColorToGray module to divide the image into the R,G and B channels and work with those individually. You may be able to use the Unmix module to distinguish bone/non-bone, AppyThreshold to produce a binary image of the non-bone region, then use MaskImage on the color channel of your choice. That way, if you use the output of MaskImage as input into a later Identify modules, it will identify features only on the non-bone regions. You could also upload an image for us to take a look at.

Hope this helps!
-Mark

Thank you Mark, I am going to try this and let you know!!

Hello Mark,

I am sending you the pipeline and two images. I think my pipeline improved but I am still recognizing some bone and adipocytes (white round areas). I want to know the area ocuppied by the stained cells (AEC+) wiht respect to all area. The problem is that the hematoxylin recongnized just nuclei and the positive cells are stromal cells very elongated. Please, can you check my pipeline? Thank you very much!!!

kena


area occupied final.cp (9.2 KB)

Hmm, these images are much more difficult since the bone is approximately the same hue as the rest of tissue that you want to include. Is there any way to stain the bone with a different color?
-Mark

Unfortunatley I can not, is there any hope??

I’m attaching a new pipeline. This attempts to detect and mask out the bone if any. The key here is relying on the fact that (a) the bone is lighter than the nuclei and (2) the bone consists of regions of large size.

You will most definitely need to optimize parameters but this should get you started.

Regards,
-Mark
2011_01_06.cp (17.5 KB)

Thank you very much for all your help! the pipeline you sent me is working very good.
kena