Re: Negative Controls
I think normal serum or isotypic-matched Ig's are useful for investigations of new antibodies and/or research studies, but they are not practical for busy routine IHC Labs. We use only diluent for our negative controls. I am more interested in identifying sources of "non-specific" staining (e.g., endogenous biotin) due to the detection system than I am with the primary antibody. In fact, I can't remember ever seeing "non-specific" staining due to the primary antibody that would be identified by using non-immune serum on the negative control (as long as the primary antibody was properly titrated). I think the majority of antibody suppliers are doing an excellent job in providing us with quality primary antibodies for routine clinical IHC. In my opinion, the use of a negative control today is less important than it was in the past because of the current availability of sensitive, one-step, non-avidin/biotin detection systems.
>>> "Philopena, Jennifer" 10/02/01 02:29PM >>>
I have two rather lengthy issues I hope some of you will be able to help me
There's been ongoing discussion (5 years) in my department about IHC
What do you use?
I've been using what seems to be the most logical to me - normal IgG's from
the same host, at the same concentration as the primary antibody.
One researcher here uses normal IgG's, but titer's it to a concentration
that doesn't give background - usually 1ug/ml - regardless of the
concentration of the primary. I don't see the point of this. Another
researcher uses just antibody diluents - here we use 0.1% BSA.
I think using IgG's controls for protein binding (non-specificity), and
using only diluents controls for any background staining not due to the
antibodies (endogenous biotin or peroxidase).
My second issue is one regarding an IHC protocol that's giving me specific
false positive staining. I'm looking at hexon staining in human tumor
xenographs in nude mice. I have a goat polyclonal and use goat IgG's as a
control - both at 10ug/ml. I usually get distinct hexon staining in tumor,
but always get some positive cells at the periphery, looks like in adipose
tissue, in both primary and control antibody sections. I omitted the
antibodies and it looks like the binding is coming from the secondary swine
or rabbit anti-goat. So i tried a mouse adsorbed secondary, and I tried
adding 2 to 10% mouse serum to the blocking step and to the secondary
antibody dilution. No consistently helpful results - my false positive (or
background) did not go away. What should I try next? The problem isn't a
bad one - it's just bothersome.
Thank you all for your thoughts.
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