refining a statement on cytokine staining, sorry

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From:Gayle Callis <>
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First, antigenic not antigenis

and other detergents MAY interact, 

Sorry about that - 

>Date: Thu, 06 Jul 2000 22:06:16 +0000
>From: Gayle Callis <>
>Subject: Re: cytokine IHC again
>Saponin is used to permeabilize the cell membrane to permit the antibody
>penetrate to intracellular cytokine antigenis sites on the Golgi.  IF you
>use saponin, you must use it from start to finish, to keep membrane open
>UNTIL just before the chromogen step, you reverse the permeablization with
>pure PBS and then add chromogen.  Rationale is saponin has a higher
>affinity for cholesterol in membranes, and will "intercalate" the membrane,
>replacing the cholestrol, which is a reversible permeablization step.
>Other detergents interact with cytokines, change structure and make the
>antigenic site unaccessible.  Also the cytokines could be solublized by
>other detertgents.  Anderssons, Sander and Litton have discussed this
>extensively in the literature.  Histonet archives, back a couple of years,
>with extensiuve discussion of detergent use including some wonderful
>explanations of saponin, basically nasty stuff!   
>Some people have successfully stained cytokines using Tween 20 (Caetano e
>Sousa, J Exp Med) with frozen sections as saponin eats frozens fixed with
>acetone, the sections are lost or look terrible.   That is why Andersson,
>Litton et al, R & D systems, etc advise using formalin or PFA fixation.
>Not so great when you want to do a CD marker (4 or 8) with IFN or IL4
>double staining.  These murine CD markers are not fond of formalin in any
>form.  Litton Am J of Pathology did an unfixed section (mouse) frozen
>stained with CD4 or 8, then fixed and proceeded with double the rest of the
>way, a bit tedious. 
>Question:  have you done a ligand blocking control to insure that your
>cytokines are or are not staining?  That seems to be an important test,
>since many people are having trouble with background staining from the
>isotype matched control that LOOKS very much like positive cytokine
>staining on tissue sections, particularly with the high concentrations of
>monoclonals often needed to achieve staining (sometimes 10ug/ml, even
>higher!)  R & D has affinity purified polyclonals that are working, but are
>They have all their protocols on their website --
>Marcia Bentz has done murine cytokine (not IL4 or IFN gamma, per her info)
>on FFPE/saponin in the buffers, successfully. Very nice work on several
>At 08:33 AM 7/14/00 -0700, you wrote:
>>To any cytokine stainers:
>>Last week put out a kind of theory question (as opposed to purely
>>procedural) re: use (or necessiity) of saponin in IHC washes on frozens and
>>paraffin when looking for cytokines.  Don't know if it made it out on the
>>Net as I've not seen a single response.
>>Have talked to people who say it is essential.  My cytokines seem to stain
>>with or without saponin.  Any ideas on the reasoning behind the saponin?
>>Thanks for any thoughts.
>>Immunex Corp.
>>Seattle, WA
>Gayle Callis
>Veterinary Molecular Biology
>Montana State University
>Bozeman MT 59717-3610
>406 994-4705
>406 994-4303
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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