Analyzing Alcian Blue Staining on IHC images

Sample image and/or code

  • Upload an original image file here directly or share via a link to a file-sharing site (such as Dropbox) – (make sure however that you are allowed to share the image data publicly under the conditions of this forum).
  • Share a minimal working example of your macro code.


  • This is an slice of a mouse submandibular gland and it is stained with Alcian Blue, an immunohistochemical stain for mucin and mucin-like substances.

Analysis goals

  • What information are you interested in getting from this image?

I am interested in quantifying the area of the blue staining while excluding the nuclei that seemed to have stained purplish.


  • What stops you from proceeding?
    When setting the threshold to detect the blue area I am not able to exclude the nuclei from the detected area.

  • What have you tried already?
    I tried the analyze particles size exclusion function used when counting nuclei but it just ends up excluding the larger blue stained sections I am actually interested in quantifying

  • Have you found any related forum topics? If so, cross-link them.

  • What software packages and/or plugins have you tried?
    I am using FIJI so it has packages installed already and I haven’t tried any outside packages yet.

1 Like

If you use FIJI or if you have the ColorDeconvolution" plugin installed then you can use the following macro command to separate your stains:

run("Colour Deconvolution2", "vectors=[User values] output=8bit_Transmittance simulated cross hide [r1]=0.78000 [g1]=0.61000 [b1]=0.136 [r2]=0.093 [g2]=0.955 [b2]=0.28 [r3]=0.00000 [g3]=0.00000 [b3]=0.00000");

to create images like this


1 Like

Hi, I think you meant “histochemical”, Alcian blue is not “immunohistochemical”.
You could try using the colour deconvolution2 plugin to unmix H, E and Alcian blue dyes and then segment the Alcian blue channel.
You will probably have to define the vectors for the 3 dyes, as the built in vector in the plugin does not include Eosin (which I think it might be the pink dye). Not sure what the very bright dots are (erythrocytes?) but it you used 4 different dyes, the method won’t work).

@phaub You beat me to the post :slight_smile:


Thank you! So I’ve been able to get this far and I try setting the threshold on the blue image and I can’t seem to exclude the nuclei from being counted in the area of the stain. I can upload a screen shot of what I mean in a while when I get back to my workstation.

Thanks you’re right! Lol it is histochemical staining