blocking Fc receptors?

Clive Gray (CLIVEG@niv.ac.za)
Wed, 19 Apr 1995 14:18:13 GMT+0200

I hope someone can help!
We are trying a saponin permeabilization technique - to measure
intracytoplasmic cytokines and other proteins (p24 etc). We are
initially fixing with 4% formaldehyde and then coming in with saponin
and then obviously labelling with our Ab. However, when we label
fixed cells only there are very high backgrounds, even though our
isotypic controls are negative. I cannot believe that vimentin
(our positive control) resides on the surface of cells (PBMC). Does
this suggest that the monoclonal Ab is binding to Fc receptors? This
is also true for other monoclonals we are using - especially when
staining in vitro activated cells. We have scratched our heads and
tried blocking with goat, rabbit or horse serum before labelling as
well as tritating out our primary Ab. Does anyone have any experience
or ideas? Are we barking up the wrong tree? I would appreciate any
comments or ideas.

Thanks

Clive Gray PhD
University of the Witwatersrand
Johannesburg, South Africa

e-mail: cliveg@niv.ac.za

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