We have a user with a nasty problem that we haven't been able to dent.
They are trying to do some immunocytochemistry on HeLa cells growing on
glass coverslips.  They fix in either acetone or 2% paraformaldehyde.  The
paraformaldehyde fixed monolayers are subsequently permeabilized with
acetone, triton x-100 or saponin (problem is the same regardless).  At this
point there is no autofluorescence at any wavelength.  When they incubate
the cells in a high quality donkey anti-mouse IgG coupled to Cy5 (Jackson)
there is a very high level on non-specific adhesion.  We have tried the
following blocking agents:  normal Donkey serum, BSA, gelatin, non-fat dry
milk, or all of the above simultaneously. Pretreating the sections with the
blocking agents has no effect.  Using other anti-mouse antibodies coupled
to FITC or rhodamine or a Goat anti-rabbit serum give the same problem.
I should point out we routinely use these antibodies in my own laboratory
following the same protocols and do not get any background binding.  We get
the same result when we personally run up the cells so we can't blame it on
the technician.  Is there something weird about HeLa cells or something
else I am missing?  Do HeLa cells have IgG receptors?  Your helpful advice
will be gratefully received.
 
 
 
Thomas E. Phillips, Ph.D.
Associate Professor of Biological Sciences
Director, Molecular Cytology Core Facility
3 Tucker Hall
University of Missouri
Columbia, MO 65211
(314)-882-4712 (voice)
(314)-882-0123 (fax)