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Control for primary antibody in Immunohistochemistry - What is it? (Aug/12/2008 )

I don't know how much of the fluorescent staining in my immunohistochemistry slides is resulted of non-specific binding of primary antibody. I know it's easy for the secondary antibody; we just eliminate the primary antibody from one slide and see how much staining we're gonna have then. But for the primary the only confident way is to test it on the exact same tissue which is only different in the expression of that particular protein (its gene is knocked out for example). I don't have such a tissue and I can not prove to people how much of my staining is specific ... I have played around my staining protocol a lot trying to remove non-specific staining as much as possible but still I'm not sure how much of it is there ...


It's a pretty expensive way, but you can always stain with just your primary antibody and don't include the secondary antibody. All the staining that comes up is background. Usually though people suffice with just the secondary antibody control.
You can also try maybe a different tissue culture cell line that is not expressing that protein. But that depends on what your staining for.


How do you do your staining?

Do you first block with serum from the animal in which the secondary is raised?
Do you control for primary antibody by using an IgG control and then come in with the secondary as usual?
I tend to avoid the no primary antibody route as a control as it doesn't really tell you anthing. If you have Fc receptors on the tissue you're going to bind antibody regardless of it's specificity and is more convincing that your staining is real.

Are you looking at a tissue such a liver? Is the background coming from something such as bile salts?
Is your antibody a polyclonal? Is there a monoclonal available that might be more suitable?
Can you buy a similar antibody from a different supplier?

-Chris W-

My antibody is a polyclonal; I have already tried another monoclonal which has not worked for this specific animal (locust) and there is no more money and time to try any other antibody; there might be better ones too. This antibody at least has worked for western blot ...
I do block my tissue in a serum from the same animal in which secondary is raised ... and I'm staining the ganglion in which the connective tissue around the ganglion is fluorescing much more than the other parts neurons themselves are stained with less extent, their nucleus is not stained ( which I'm at least happy about this part ) but there is some level of staining everywhere which makes me worried about non-specific binding of antibody ... I'll attach a picture of my stainings ...
and you are also talking about an IgG control, I don't understand what it is ...


Some one has suggested me using the pre-immunized serum of the same species in which the primary antibody has been raised in my case rabbit. So we should do the same procedure as we do for our normal immunostaining except instead of primary antibody we incubate the slide in this pre-immunized serum, has anybody done that? Do you think it could be really a control for the primary antibody specificity?