Immunohistochemistry--How to quantify it? - (Nov/25/2008 )

Hi all, I am trying to quantify my IHC staining (with haemotoxylin counter stain) picture via scoring 5 fields base on their intensity. This is very labouring and inefficient method from my viewpoint. I read other papers that they quantify it with ImageJ or Image pro. We have both software in the institute but no one has actually use it for quantifying IHC. I really appreciate if anybody has any protocol available for this application.

Thank you so much!!

OT

I would say that this sort of thing is very very tricky and inherently flawed unless you can standardize your quantitation in some manner. If you are doing your IHC by hand, then forget trying to quantitate as differences between runs will be quite large depending on a number of factors such as freshness of solutions, times used, wash steps, antibody storage etc.

If you are running on an immunostainer then you could attempt it, but you have to think about your controls very carefully and whether or not what patterns you get could be detected by eye and by an independent observer who doesn't know what you have stained for.

Also what does your staining pattern look like: is it whole cell, nuclear, perinuclear? How will your quantitation method work for what you are trying to do?

Sorry, we don't have a protocol for it, we have a resident pathologist who grades all our staining.

Anyway, try the ImageJ webpage, there is a lot of stuff there!

Hi, thanks for the reply. I agree with you that each experiment run do produce slight variation. What I am doing now is just trying to compare slides within each experiment. we have 6 patient slides (with different disease conditions) for each experiment. Some conditions give me nucleus staining while others don't. I am planning to quantify both via staining intensity and the number of nucleus staining. Our previos scoring system was done by a blind observer.

I went to ImageJ website before and it turns out I don't really understand at all (these things are really hard to understand).
Anyway, thanks for your opinion and have a nice day!