Re: Measuring protein expression intensity

Posted by Demilux on Mar 05, 2014; 6:17pm
URL: http://imagej.273.s1.nabble.com/Measuring-protein-expression-intensity-tp5006771p5006788.html

Thanks for the reply.

What bothers me is that everyone is suggesting the colour deconvolution plugin, which is used when one needs to separate colours/stains in staining (from what I got). What I need is just to quantify the intensity of a BW image, and I can't seem to use the plugin for that (maybe it's possible, but I don't know how to properly use the plugin). If this is possible, would someone be kind enough to explain how?

Another thing which I fail to understand (I'm new at this, don't take it hard on me) is why exactly "quantifying" bright field IHC staining is considered unreliable. The brighter the colour, the less protein there is expressed, and vice versa. So, why is it a bad idea to assign relative numbers to the intensities (0 = black = max protein expression; 255 = white = no protein)? I would use the same light intensity settings on the microscope/camera for each plate and then compare them one to another. As I said, someone suggested using histograms. But, if this is such a bad idea (?), do you have any other suggestions?

Thanks ahead