I read your e-mail and thought I should respond. In your first question you are absolutely right if you use a polyclonal antibody your isotype control is a polyclonal IgG from the same host, at the same concentration as the primary antibody. You can not do a lower concentration of your isotype to avoid background that is just plain wrong. You can use the isotype at a higher concentration if you want to save slides. I had two antibodies with the same isotype one worked at 1ug/ml and another at 6.7ug/ml, I only did slides at 6.7, if it doesn't bind there it won't bind at 1ug/ml. If the antibody is monoclonal I always use the specific Isotype example: Rat IgG2a...not just rat IgG. Also using a PBS or BSA instead of the isotype is just a control for the system (peroxidase, avidin,biotin, and IgG secondary binding to B-cells). It's a good control if you think your system is causing background.
Your second question is one I've seen in my mouse tissues as well. sometimes in mast cells or adipose tissues. I eliminated everything one at a time and it came down to the streptavidin peroxidase. Short of changing my system to a non-peroxidase based system I just deal with a little background that I can explain away. Hope this helps.
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>>> "Philopena, Jennifer" 10/02 2:29 PM >>>
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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