Marcia,
            I was trying something similar with murine spleen treated with LPS and trying to stain for cytokines (IFN-gamma, TNF-alpha), all my attempts failed big time.  I have since given up for the time being on cytokine staining. The only articles I've seen that our pathologist and I believe is by a Swedish gentlemen Mark J. Litton that has done lots of cytokine staining. He was also on a paper that was looking at cytokine staining in a infectious disease model, that had boat loads of staining. I believe this is the way to go ,you may need lots of cytokine around to see it by immuno.  I have not been able to reproduce any cytokine staining from their protocol that does not have lots of isotype control background. I give up for now and maybe later will find new inspiration. As far as paraffin goes if you can't get it to work in frozen I wouldn't waste your time with paraffin samples.  Sorry to sound so negative but I've been trying cytokine staining  off and on for 4 years !
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or more with no concrete positiv
e results. Maybe Gayle will have some new ideas. I lot forward to any and all responses you may receive. 

Jamie Erickson
Genetics Institute
Andover, MA 01810

>>> Marcia Bentz <mb7x@virginia.edu> 08/18 7:45 AM >>>
Hi,
Just me again pulling my hair out with the cytokine staining on mouse
kidney. Still getting inappropriate signal on negative control (frozen
sections), but it seems to be specific for tubular epithelial cells. Is
there anything special about these cells that would make them "sticky" with
an ABC peroxidase kit? Interestingly, the kidney treated with LPS seemed to
show significantly less of this than the untreated kidney.

Also since morphology pretty much sucks on frozens after all the processing
and detergents, I would prefer to do paraffin embedded. Is there a better
fixative than 10% buffered formalin for cytokine IHC? And is it likely that
a citric acid buffer in a 90 C waterbath will work for antigen retreival?
(help Gayle!)

As always, all hints and suggestions are welcome!

Thanks for always helping,
Marcia

Lab Specialist Sr.
Univ. of Virginia
Division of Nephrology


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