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On a different subject:
Several weeks ago, someone mentioned blocking the binding of Ig to Fc
receptors on mouse cells using antibodies specific for the FcR.
Pharmingen sells Fc Block for this purpose, but does anyone know of an
antibody available to block these receptors on rabbit cells? We are
having a problem with an immunofluorescence assay that involves rabbit
myocytes and it appears that non-specific binding of rabbit Ig to
these cells may be the source of the difficulty.
The assay involves demonstration of protein kinase C in rabbit
myocytes using a rabbit anti PKC antibody [we recognize the problems this
could create but so far the investigator hasn't found an anti-PKC antibody
that works as well as this particular one made in the rabbit].
This particular anti PKC (prot. kinase c) antibody is only available as
serum - neither purified Ig, affinity purified Ig, nor F(ab)'2 fragments
are available.
The myocytes are freshly isolated from rabbit hearts, deposited on slides
with a Cytospin, fixed with
-20 C methanol for 5 min, blocked with BSA or normal goat serum (this is an
indirect assay and the 2nd Ab was made in a goat) for one hour at RT and
then labelled with (ultracentrifuged) anti PKC followed by ultracentrifuged
FITC-conjugated, goat anti rabbit Ig.
The 2nd Ab alone (goat anti rabbit-FITC) does not stain the cells. Some
non-specific staining does occur with normal rabbit serum followed by
the FITC-conjugated goat anti rabbit antibody. Although it is not as
intense as, nor does it have the same pattern of reactivity as does, the
rabbit anti PKC antibody, we would nevertheless prefer to lower this
binding and its fluorescence.
It looks as if FcR may be playing a role and since we don't have
F(ab)'2 fragments of the anti PKC, we can't test this (the expense of even
small amounts of the commercially available anti PKC is too great to
consider making these fragments from this antibody).
Does anyone know a source (commercial or individual) of anti rabbit FcR
(made in something other than a rabbit)?
If so, we would like to determine if it blocks non-specific binding of
rabbit Ig to rabbit myocytes.
Thanks,
Ray
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Raymond B. Hester, Ph.D. Someday, after we have mastered the
Flow Cytometry Lab winds, the waves, the tides and
The University of South Alabama gravity, we shall harness the energies
Mobile, AL 36688-0002 of love. Then, for the second time
Email: rhester@jaguar1.usouthal.edu in the history of the world,
Voice: (334) 460-6029 man will have discovered fire.
FAX: (334) 460-6073 -Teilhard de Chardin
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