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.

     Ronnie Houston
     Regional Histology Operations Manager
     Bon Secours HealthPartners Laboratories
     5801 Bremo Road
     Richmond, VA 23226

______________________________ Reply Separator _________________________________
Subject: Ag retreival vs. Permeabilizing
Author:  Greg Dobbin  at BSHSIBTW
Date:    8/6/02 12:54 PM

       Hello All,

       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??
       Greg Dobbin
       Pathology Lab
       Atlantic Veterinary College, U.P.E.I.
       550 University Ave.
       Charlottetown, P.E.I.
       Canada,  C1A 4P3
       Phone: (902)566-0744
       Fax: (902)566-0851
       "A farmer is a person outstanding in their field."

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