Cell permeabilization
Clive Gray (CLIVEG@niv.ac.za)
Thu, 11 May 1995 15:31:54 GMT+0200
Hello everyone. I would like to thank all people who replied to my
request re blocking Fc receptors using a saponin permeabilization
method (I'm a bit late in responding as our net has been down - can
you believe). I had very good feedback with some very useful comments
and hints indeed. Some of the comments included diluting out the
primary and secondary antibodies - aiming to reduce background
signals; using fab or fab'2 fragments of our primary monoclonal; using
mouse IgG2a to block and formaldehyde fixation itself is helping to
permeabilize cells. We have looked at the latter suggestion in more
detail and sure enough at 4% concentration (without introducing
saponin) we were getting well permeabilized cells (using PI in
conjunction with anti-vimentin). We have shown that at about 1.5%
formaldehyde at a 15min incubation, we get quite high PI staining. We
have also decided to used purified human IgG to further block any
potential Fc receptors (especially on activated cells). At least I
am beginning to get to the bottom of my high "backgrounds". Thanks
again.
Clive Gray PhD
University of the Witwatersrand
Johannesburg
South Africa
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