Re: Blocking Fc receptors on rabbit myocytes
Nicholas King (nickk@med.su.oz.au)
Fri, 18 Aug 1995 13:32:43 +1000
Ray Hester writes:
>
>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.
>
There are a number of different ways that you could tackle this. I don't
really think that it is likely to be Fc receptors in myocytes, since your
second Ab doesn't label these cells. We have never been troubled by
non-specific staining on myocytes that have been surface labelled and not
fixed, although we have never looked in the rabbit.
One thing you could try is a change of fixative; acetone 100% -20 deg. C
FOR 15' is a good fixative for cytospun slides and is unlikely to result in
loss of antigenic configuration, SO LONG AS YOU DO NOT ALLOW THE SAMPLE DRY
- you can rehydrate it with PBS after the slides come out of it.
The other thing is a simple Protein A column will get the anti-PKC antibody
out of the whole rabbit serum very quickly and well (unlike from mouse
serum); you would then be able to clean up the Preimmune serum in the same
way to reduce N/S labelling.
Lastly, a quick and simple way of reducing N/S labelling is to use some
detergent in the Ab solution and the washes about 0.1% for starters, but
titrating it would be best. In addition, or separately, you could also use
an excellent reducer of N/S staining from intracellular sources: Dextran
Sulphate at about 0.1% in the washes.
I hope this helps.
Regards,
Nicholas King
Nicholas King
Department of Pathology
Blackburn Building, D06
University of Sydney
New South Wales 2006
Australia.
nickk@blackburn.med.su.oz.au
ph.61 2 351 4553
FAX: 61 2 351 3429