Macro for IHC quantification

Dear team,

I’m trying to analyze histological samples stained with a specific antibody, that brings a brown color (typical immunohistochemistry). In this case, renal tissue.
I found in the literature a protocol for easy quantification of this kind of images (Semi-quantitative Determination of Protein Expression using Immunohistochemistry Staining and Analysis: An Integrated Protocol - PubMed), and when I tried with one picture, it worked fine. The problem started when I aimed to produce a macro for an automatic (and quick) analysis of 140 images. When I recorded the procedure, it took the name of the image, that I don’t know how to eliminate from the macro, so that the program analyses all pictures of the input folder. In addition, after color deconvolution, only channel 2 is the correct one to measure, but I also don’t know how to select it, instead of channel 3.
Here is the macro I have after recording (sorry, I don’t know how to attach files here):
run(“Colour Deconvolution”, “vectors=[H DAB]”);
selectWindow(“getIT_1.tif-(Colour_3)”);
selectWindow(“getIT_1.tif-(Colour_2)”);
setAutoThreshold(“Default”);
//run(“Threshold…”);
//setThreshold(0, 190);
setOption(“BlackBackground”, false);
run(“Convert to Mask”);
run(“Measure”);

I would appreciate it a lot if you can help me here. Maybe it’s only a tiny thing, but I’m unable to find the solution alone!

Cristina

Hi @crismhigueras,

Please be aware that the publication you linked is not describing a suitable procedure for DAB quantification or similar histological staining.

To enable people here to further help you, it would be good if you can provide one of your images as an example (also possible as a link to a cloud storage).
Furthermore, it would be necessary that you describe your needs as detailed as possible.