I'm going bald also! and all the new growth is coming in totally gray,
have started to call this project "Cytokines from Hell!", sorry friends
and colleagues, but I have been swimming the River Styx daily on my way
into the throes of Hades.

Marcia

	You didn't say which cytokine you were working with, but should
that make a difference??? Hopefully your LPS stimulation is causing the
cytokine to be expressed.  

Unfortunately, the norm for sections seems to be frozens for most people.
I have IL-4 up and running, with an appropriate control today, both for
immunoperoxidase and IFA, to see if we can get optimal results, finally.
WE have a tissue that is expressing IL-4 quite well, and should as 
predicted.

Frozens, air dried a LONG time, 4 hours or more, if possible, cold 4C fresh
acetone fix for 10 min, air dry for 1 hour, frozen down at -80.  The buffer is
TBS with 0.05% Tween 20, pH 7.4, peroxidase block is Dako for 15 min.

Target concentration is all over the place for monoclonals only, with
an appropriate IgG isotype matched control.  ug/ml seems to vary from
5 up to 20 ug/ml, which is a bummer with the control IgG. I have not
done any blocking with normal serums or BSA unless I do saponin 
permeablization on PFA fixed sections.  I use the purest of pure,
heat inactivated serum, microfiltered, and BSA from Jackson, immunoglobulin
and protease free, also microfiltered after making up.

So one protocol is TBS/Tween with perox block MEOH free, dilute antibody in
buffer, secondary is mouse antirat IgG F(ab')2 biotin, SA HRP, and
Pierce DAB enhanced, very sensitive which is what you need for 
the intracellular cytokine staining.  This is also filtered.  There are no
normal serum blocks with this method, and it is relatively clean, the
background seems to be higher on negative control vs the target tissue,
and I think this is a binding of high concentration of Isotype IgG to
tissues, no specific cells, and on the outside of tissues/cells since you have
to have such a high concentration of antibody to stain the cytokine.  
Things to try, dilute your secondary to not give background, but still stain.
AND try 36C incubation during day, or overnight in refrig.   When I did
normal serum blocks, etc, I had so much background, I gave up since the
secondary works well, plus the Tween for blocking.  It is a start. 

If you want to do double stain for other cell markers, the acetone becomes 
a fixative of choice for mouse.  However, you can do fresh formalin in
PBS, 4C for 10 min (NO ANTIGEN RETRIEVAL FOR CYTOKINES).  You should access
Pharmingens cytokines for cells staining, it is basically the same thing
you need to do for tissue sections.  They have matched some of their
isotype controls to their monoclonals, haven't quite figured out why
any IgG isotype control wouldn't work, as some of these are cheaper from
other sources.  Their monoclonals are excellent, and the literature
has given us many clones for cytokines that work for IHC. 

If I do saponin on frozens fixed with acetone, I HAVE TO USE THE 
INSTRUMEDICS TAPE TRANSFER TO RETAIN TISSUE MORPHOLOGY. Have a love/hate
relationship with saponin, chews on frozens except for formalin fixed
frozens unless the section is glued to that slide with polymer.  Our
dilemma is doing T cell markers after cytokine, and formalin
shoots that one down.  I have decided to try and avoid saponin, two other
labs did so, with excellent results.  

I have gone to daytime incubations, upping it to 37C today, to try and speed
things along.  My first IL-4 run was good after many hours at RT, day run.

What color wig are you buying?  I could be a blonde for the first time in
my life! and hopefully have more fun than staining these cytokines.

Gayle Callis

Oh well, 

Need I say more, but progress is being made at least with one, if
that succeeds, then the next will be in lineup.