I’m attempting IHC on human DRG/nerve using human serum (containing putative anti-human antibodies).
The issue I’m running into is that there is huge background with just the secondary alone (goat anti-human IgG).
How do people get around this? I know Mouse-on-Mouse background is a thing, but honestly I’ve never seen it despite using lots of mouse antibodies on mouse tissue.
But this human background issue is real. Any thoughts? @sshiers
These are some possible solutions:
https://vectorlabs.com/human-on-human-immunodetection-kit.html
https://www.jacksonimmuno.com/technical/products/groups/fab/blocking
Hmm this is going to be challenging. So Human serum with anti-human antibodies in it? How does that work, are these autoantibodies?
The background issue is because your anti-human secondary is binding to all of the IgG that are innate to tissue. Its not perfused, so all those IgGs are fixed right in there and since the DRG seems more vascularized in human than mouse, its probably an awful amount of background!
In mouse, the mouse antibody issue is a problem if you are using non-perfused highly-vascularized tissues. That’s why most people perfuse, to get rid of all the IgGs in the blood… in brain it can be a problem, but its not as noticeable in the DRG. If I am using non-perfused mouse tissue, I cut the background down by using isotype specific anti-mouse secondaries since most mouse primary antibodies are monoclonal. But you cant do that for human since I assume your preps are polyclonal.
That kit from Vector labs seems promising - looks like they “block” all the innate IgGs with some type of human primary. You have to use immunoperoxidase staining though so you are limited to just one primary.
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Great points @sshiers
Yes, I think the issue is the endogenous IgG.
I am going to try the Fab blocking method
https://www.jacksonimmuno.com/technical/products/groups/fab/blocking
Basically overwhelm the IgG with unlabeled Fab to block all the endogenous IgG. Then apply you primary/serum, which should bind to the still-exposed target antigen sites.
Yes, I’m looking at autoantibodies in human serum.
Will let you know how it goes