DAB staining

I’m trying to use Cellprofiler to quantify some DAB staining. my protein of interest is cytoplasmic and I would find a way to measure the intensity and compare different samples.
I have modify the simple pipeline that is given at the beginning, but I’m not sure if the results is correct…in particular I’m not sure if I have to convert all the channel to gray or only the red one…

Thank you for the help

testDAB.cp (9.52 KB)

Hi Sara,

I think your pipeline is on the right track. I’m attaching a modified pipeline that unmixes the colors (as you’ve already done), identifies the tissue region and measures the DAB intensity against the identified tissue object.

As an aside, it’s usually preferable to use TIFs or PNGs for analysis since they don’t lose information. I think you might be doing this already, but I want to make sure. :smiley:

2011_08_31.cp (7.07 KB)

Thank you!
using the tissue instead of identifying all the different objects is better! and easier and faster…!
in this way I can use this pipeline for every kind of DAB staining, or not?? :unamused:

and , yes I’m using .tif images…but I wasn’t able to load my image on the forum :wink:

thanks for this software!

Consistency is key here. The color unmixing method by assuming certain relative absorbances by the dyes, which makes relative color or hue of the images an important factor. If the sample illumination drastically changes or you change your staining protocol between samples, the approach may not be guaranteed to work in all cases.