Re: Ag retreival vs. Permeabilizing
My understanding of the process is that permeabilization agents act by
forming small openings or pores in cell membarnes allowing antibodies to
have access to intracellular structures in intact cells. If however, the
cells have been fixed with a solvent fixative, eg acetone or alcohol,
permeabilization is unnecessary as the fixation extracts lipid from the
cell membrane,allowing the antibodies access to the internal structures.
Permeabilization does not break down cross-links following formalin
There are differences in the action of some types of permeabilization
reagents employed in immunocytochemistry.
Triton X-100 and Tween-20 are non-ionic, non-denaturing detergents. It is
not uncommon for antibodies to not recognize their epitopes following this
type of treatment. This may be due to the fact that the antibody may only
recognize denatured protein, or because there is extensive cross-linking
around the protein of interest.
Saponin acts on cell membranes that are rich in cholesterol. The important
factor when using saponin is that all antibody dilutions, incubations and
washes should employ buffer and saponin (0.05%) Saponin-treated membranes
are not permeable to antibodies that have never been exposed to saponin in
It is important to note though that saponin will not permeabilize membranes
that do not contain cholesterol ie mitochondrial membrane and inner nuclear
Of all the permeabilization agents in common use, saponin is by far the
most gentle in its action.
I am not aware of permeabilization agents being used with sectioned
material, but only cytospins, smears, cell-cultures and whole mounts,
although I am sure someone will prove me wrong.
Immunocytochemical Methods and Protocols, 2nd edition, ed Javois LC, Humana
Press 1999, ISBN 0-89603-570-0 provides much more detail than given here.
Regional Histology Operations Manager
Bon Secours HealthPartners Laboratories
5801 Bremo Road
Richmond, VA 23226
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Subject: Ag retreival vs. Permeabilizing
Author: Greg Dobbin at BSHSIBTW
Date: 8/6/02 12:54 PM
I found a "Brief Communication" in Vet Pathology (38:116-119; 2001) that
discusses expression of COX-2 in canine renal carcinomas. The authors do
not mention antigen retreival,
they speak of permeabilizing the tissues using triton and saponin. Is
permeabilizing (as described) a form of antigen retreival or does it
serve another purpose?
Has anyone else heard of this before? Explanation??
Atlantic Veterinary College, U.P.E.I.
550 University Ave.
Canada, C1A 4P3
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