RE: human-human ihc

From:Carlos Genty

Hi Steve:

I think Rob is correct.  I would add though that you can try an approach
similar to a paper in the Journal Histochemistry and Cytochemistry*.  I
believe that this approach as also used in Dako's ARK kit and a few other
vendors.  There is no reason for this not to work on Humans.   You will need
to purchase a conjugate anti-Human (Fab specific) secondary antibody and
some human serum.  Complex the primary and secondary in a tube first (15
minutes or longer if you wish).  Then block unbound Human IgG's with normal
human serum for 5 to 10 minutes (in the same tube obviously). You can then
apply the complex to the tissue without any specificity issues.  Then detect
as usual, depending on your preferred conjugate.  I have been doing this for
some time now, albeit in the mouse world, and it works great.   The drawback
to any of these techniques is sensitivity.

These labeled Fab fragments can be obtained from Sigma.  Of course you will
need to set up the appropriate titration experiments.

*Here is the abstract:

A novel modification of the avidin-biotin complex method for
immunohistochemical studies of transgenic mice with murine monoclonal
KM Fung, A Messing, VM Lee and JQ Trojanowski

Department of Pathology & Laboratory Medicine, University of Pennsylvania
School of Medicine, Philadelphia 19104.

When mouse tissues are probed with murine monoclonal antibodies (MAb) by
indirect immunohistochemistry, the secondary antibody detects tissue- bound
MAb and irrelevant, endogenous mouse immunoglobulins. The latter are a
source of confounding background, especially in diseased tissues. To
circumvent this problem, we generated complexes of primary MAb and
biotinylated secondary antibodies in vitro for use as antigen-specific
probes. After blocking free binding sites in the complexed secondary
antibodies with normal mouse serum, the complexes were applied to mouse
tissue sections and tissue-bound complexes were visualized with an
avidin-biotin detection system. Complexes formed with 12 different rat or
mouse MAb were used to probe sections of normal mice, tumor-bearing
transgenic mice, and mice with tumor xenografts. The staining patterns
produced by these probes reflected the specificity of the MAb in the
complexes, and the labeling of irrelevant, endogenous mouse immunoglobulins
was reduced substantially. This novel, indirect immunohistochemical method
can be exploited to study normal and diseased mouse tissues using a variety
of murine MAb.

I hope this helps!

Carlos Genty
Breast Center Pathology
Baylor College of Medicine
Houston, Texas

-----Original Message-----
From: Robert Geske []
Sent: Monday, December 17, 2001 6:11 PM
To: 'Crochiere, Steve '; 'Histonet (E-mail) '
Subject: RE: human-human ihc


you're singing the same blues tune we who routinely work with non human
animal tissue sing ---- just a different verse.  i would suggest purchasing
the antibody conjugated to some reporter (biotin, peroxidase, fluorochrome,
etc) and remove the need for the secondary antibody.  if the antibody is not
available conjugated you could purchase a kit and do the conjugation


-----Original Message-----
From: Crochiere, Steve
To: Histonet (E-mail)
Sent: 12/17/01 11:53 AM
Subject: human-human ihc

Does anyone out there have an IHC procedure for staining human tissue
human Mab's? I have tried to do this by blocking endogrnous peroxidase
H2O2,  IgG activity with a FAB fragment, protein with normal serum, and
biotin with the vectorlabs biotin blocking sysytem, and still have so
background and non-specific staining in the negative controls (IgG whole
molecule) that the slides are unreadable.
Any suggestions would be greatly appreciated.

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