Using Human antibodies for IHC on HUMAN tissue
|From:||Miriam Schroeder <firstname.lastname@example.org>|
Hi all. I have a request to use a HUMAN antibody as
the primary antibody for staining both mouse tissue
(shouldn't be a problem) and HUMAN tissue.
I am anticipating staining the human tissue will be
problematic due to 1)Secondary (anti-human) antibody
reacting with endogenous antibodies already in the
tissue AND/OR 2)Capture of the primary antibody by Fc
My usual procedure for IHC is to use a biotinylated
secondary, then streptavidin-peroxidase conjugate &
finally DAB. I am thinking if there were a way to
biotinylate my primary antibody (prior to application
to the tissue) that I could avoid using the
biotinylated secondary & get around cross-reactivity
problems that way.
I am wondering if anyone has a tried-and-true
procedure for dealing with this sort of situation.
(Or, preferrably, knows of a kit for biotinylation of
the primary HUMAN antibodies.) (I've seen lots of
kits for biotinylating MOUSE primaries, but haven't
come across any for HUMAN primaries.)
I realize there may be other ways around this, like
"block" endogenous antibodies in the tissue with an
UNLABELED anti-human secondary at the beginning of the
procedure, pre-adsorbing the secondary (but I'd like
to steer clear of human serum, don't know where to get
it anyway), etc. etc. Time is of the essence so I
don't have time to work out a procedure on my own that
might employ these type methods.
Any help from someone with ACTUAL EXPERIENCE in this
type situation would be greatly appreciated. We can
all hypothesize about ways around this (see above
paragraph) until we are blue in the face but at the
end of the day any such procedure would have to be
developed/optimized first, and I don't have time for
Thanks so much!
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