cytokine staining on LPS treated mouse kidney

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From:Marcia Bentz <>
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Hi all,
Still working with the same darn mouse kidney and cytokine staining
exclusively. I've managed beautiful stains on FFPE for IL1 beta, IL8,
macropahges, neutrophils, but my boss really wants results for IL6 and TNF
alpha. I actually have done great TNF staining on a TNF transgenic pancreas
(FFPE), but our experimental kidneys show nothing, even though the presence
of these cytokines is evident by ELISA and RPA analysis.He wants to go back
to trying frozens sections, but last year when we tried this I got
uncontrollable background, even with avidin biotin block, peroxidase block
etc. I've searced literature high and low and havn't found much and what I
have found looks pretty questionable in terms of specific staining.
My questions are:
Has anyone out there done TNF alpha and IL6 on MOUSE KIDNEY, what company
did you get the primary antibodies from (currently using PharMingen), with
frozens, any special way you prepared the tissue, ie fixed before frozen,
cryopreserved, fresh frozen than fixed with acetone, paraformaldehyde,
ethanol, embedded in OCT (this is what I've always done, fresh frozen in
OCT to -70 til cutting). 
Should I try something other than immunoperoxidase for frozen kidney?

Any and all suggestions will be greatly appreciated!


Marcia Bentz
Lab Specialist Sr
University of Virginia

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