Re: cytokine IHC again

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From:Gayle Callis <>,
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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

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