cytokine staining pattern in cells

From:"C.M. vander Loos" <>

>Gail wrote:
>Not interested in paraffin sections/antigen retrieved, only frozen
>sections, and not formalin fixed, must be acetone or acetone/alcohol
>fixation.  Interested in IFN gamma and IL4
>Two questions:
>what kind of staining pattern are you seeing with cytokine staining?
>Perinuclear staining?  Many cells grouped in a pattern
>Also, have you proven it is cytokine you have stained and not cross
>reactivity of isotype matched control to cells within the tissue, eg
>spleen?  and did you do ligand block or a specificity control to prove it
>was cytokine?


Dear Gail,
I am afraid I have a very sad message for you: forget about IFNg and IL4
staining in acetone fixed tissue sections!
We have just performed an extensive study using 13 different commercial
available anti-IFNg antibodies. We obtained some hard evidence that it is
not working: activated T-cells remain unstained and the positive signal you
get is most likely nonspecific staining of a 5% plasma cell subset. We are
very surprised that immunohistochemical staining of IFNg staining and other
soluble cytokines have been applied by so many people and have published on
it, sometimes even illustrated.
A preface to our publication can be found in the Abstract book of the XIth
Int. Congr. of Histochem. and Cytochem. last September in York, UK (pp.
72-73, poster E20: Immunohistochemical visualization of IFNg: fake or
fact?). An extensive publication is on its way to JHC.

The only thing what is reliable in this respect is performing IFNg
immunocytochemistry on intact cultured cells, preferably PFA-fixed and
saponin in all your reagents.

Best wishes, Chris

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