Myriam Marussig and cytokines

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From:Gayle Callis <>
Date:Fri, 23 Jul 1999 10:17:42 -0600

Myriam other interested cytokine groupies, this is a longgggggg one! 

>From your brief message on cytokine staining, your protocol is one that has
worked (formalin fixation/saponin permeabilization) for others.  The problem
that arises is to double stain for murine CD4 and CD8 and cytokine, plus other
CD markers. Using formalin on mouse frozen sections may work for the cytokine,
but is not compatible for some of these surface markers, even with 
overnight incubation of the antibodies.  Epitope retrieval doesn't
always work with CD4 and 8, postformalin, and I personally do not want
to deal with retrieval, which often doesn't work with formalin fixed frozens
for CD4 and 8, got an attitude about that, why bother.
I know of two groups who successfully stained cell surface markers and 
cytokines (double) using acetone fixation. That is the focus of our work and 
it looks promising.  

One thing I see on Histonet is the reference to R&D cytokine staining
procedure.  Clarification here, R&D did not do this work, it is taken from
publications by Sander, Anderssons and Litton or combinations of these authors
Methods is based on using formalin fixation of frozen sections and/or
cells with saponin.  We had no success with R&D 
polyclonals, and took Andersson/Litton advice , work exclusively with
anticytokine monoclonals, some of which we can produce from ATCC cell
lines.  One very personal opinion (what's new!) PharMingen has been
an industry standard for us, with their testing on tissue and cell
staining testing.  There are some other companies who also do this, 
bless them!  Sure saves a lot of frustration, time and money.   

PharMingen has just put out a booklet for immunoCYTOchemistry, based on same 
methods, they tweaked it, using their monoclonal antibodies (excellent) 
plus protein transport inhibitors (can't be used on frozen sections!), 
their IgG isotype control, secondaries, etc.  Results on cells are very nice.

I would be very interested in your fine tuned points of staining.  I know 
some other tricks to do cell surface markers before formalin fixation
for cytokines.  This is in literature from a publication by Mark 
Litton, but is a delicate protocol.  I know others who have had success
with various cytokines, but no protocols have been seen to date for a 
comparison to published methods, other discussion, etc..  
Would love that, and if I ever get it to work - your screens will light up 
like sunrise!

Gayle Callis.  

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