Re: Antigen Retrieval
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From: | "John A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | "Bennett, Catherine (Katie)" <cbennett@lrri.org> |
Reply-To: | |
Date: | Mon, 18 Oct 1999 14:17:27 -0400 (EDT) |
Content-Type: | text/plain; charset="us-ascii" |
>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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