Re: Antigen Retrieval

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From:"John A. Kiernan" <> (by way of histonet)
Date:Wed, 20 Oct 1999 03:46:51 -0400
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>Do all antigen retrieval procedures do the same thing?  In other words, can
>an antigen retrieval procedure that is used in one immunohistochemical run
>be used successively in another IHC run?  Or is it more a case of trial and
>error to see which antigen retrieval will work in any given new IHC
>procedure? ...  Let's say for this query that fixation and tissue type are
>the same, just primary antibody is different.

  Antigen retrieval works by causing either acid- or base-catalysed
   hydrolysis of methylene cross-links between protein molecules,
  which are introduced by fixation in formaldehyde, and possibly also
  by removing calcium ions (citrate and EDTA are common ingredients
  of retrieval solutions). Most antigens are retrieved by heating in either
  acidified (pH 1) or alkaline (pH 8-10) water, some by either or neither
  of these. Less extreme pH levels (e.g. 6) are often effective and less likely
  to cause section losses. Something about each antigen seems to
  determine which retrieval method is most effective. There are some
  good surveys in the literature, with tables showing the effects of
  different retrieval methods on many antigens. Here is an very briefly
  annotated list of 4 such papers.

   Taylor,CR; Shi,SR; Cote,RJ (1996a): Antigen retrieval for
     immunohistochemistry - Status and need for greater
     standardization. Appl. Immunohistochem. 4(3), 144-166.

        <<Review of antigen retrieval procedures for paraffin
          sections of formaldehyde-fixed tissues>>

   Taylor,CR; Shi,SR; Chen,C; Young,L; Yang,C; Cote,RJ (1996b):
     Comparative study of antigen retrieval heating methods:
     Microwave, microwave and pressure cooker, autoclave, and
     steamer. Biotech. Histochem. 71(5, Sep), 263-270.

        <<Comparative study of antigen retrieval heating methods:
          Microwave, microwave and pressure cooker, autoclave, and
          steamer. All methods effective; extended microwave and
          autoclave gave greatest sensitivity, but this could also
          be achieved by extending time in steamer.>>

   Pileri,SA; Roncador,G; Ceccarelli,C; Piccioli,M;
     Briskomatis,A; Sabattini,E; Ascani,S; Santini,D; Piccaluga,PP;
     Leone,O; Damiani,S; Ercolessi,C; Sandri,F; Pieri,F;
     Leoncini,L; Falini,B (1997): Antigen retrieval techniques in
     immunohistochemistry: Comparison of different methods. J.
     Pathol. 183(1, Sep), 116-123.

        <<61 antibodies tested. 1 mM EDTA-NaOH (pH 8) was better
          generally than citrate (6.0) or TRIS buffer (8.0) or
          protease XIV (5 m, 37C). Most convenient treatment was in
          pressure cooker for 2 min. Some antibodies better with
          the other methods.>>

   Shi,SR; Cote,RJ; Chaiwun,B; Young,LL; Shi,Y; Hawes,D; Chen,TY;
     Taylor,CR (1998): Standardization of immunohistochemistry
     based on antigen retrieval technique for routine
     formalin-fixed tissue sections. Appl. Immunohistochem. 6(2,
     Jun), 89-96.

        <<Most antigens are retrieved in formalin-fixed paraffin
        sections treated at pH either 1 or 10. One needed pH 1.>>

  The antigen retrieval solutions used in the above publications are all
  easy to make up, and there does not seem much point in buying one
  commercially, especially if it turns out to be less than optimal for the
  antigen you want to detect. Don't use any solution unless you know
  its composition.

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