Have you tried methanol fixation?  It may prove helpful.
 
>We are having a difficult time trying to stain PMNs with an antibody for
>confocal analysis. We use a primary/secondary antibody technique for
>the alpha subunit of G proteins.  Specifically, our problem is that the
>background of our secondary antibody is very high.  Our primary
>antibody is a mouse monoclonal.  Our secondary antibody is a
>FITC-F(ab)2 anti-mouse.  We have tried an intact anti-mouse IgG and it
>gave higher backgrounds, possibly due to Fc receptor binding.  We have
>tried blocking with serum (goat and human), and using low
>concentrations of triton x-100 to diseperse weak charges, yet our
>secondary antibody binds by itself with a very strong signal. Any
>suggestions?
>P.S. These are formalin-fixed cells with saponin permeabilization.
 
Sincerely,
 
Kamiar Moin, Ph.D.
Assistant Professor of Pharmacology
Wayne State University School of Medicine
Detroit, MI 48201
 
Tel:  (313)577-0514
        (313)577-1112
FAX: (313)577-6739
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