Hi Gayles ! Hi Marcia ! and all histonetters !

		Gayles and Marcia, I have been performing cytokine
staining for several years and I have have very beautiful stainings, in
different tissues (liver, brain, spleen, lymph nodes, intestinal mucosa
or isolated cells from mouse or human. 
		If you are interested to exchange " small tricks " , you
can contact me, I would be happy if I can help you. As you have done for
me trough your messages on histonet.

		Myriam Marussig
		Manager, Biological Studies
		Biovector Therapeutics, SA
		Labège, France


				-----Message d'origine-----
			De:	Gayle Callis
[SMTP:uvsgc@msu.oscs.montana.edu]
			Date:	jeudi 22 juillet 1999 18:12
			À:	histonet@Pathology.swmed.edu
			Objet:	cytokine staining

			Pharmingens haws monoclonals for cytokines, but
the immunostaining is difficult.
			Labs who have had the most success use these
clones, and it it well
			documented in the literature.

			The literature, for murine cytokine IHC, is done
on frozen sections, with
			some groups successful with formalin fixation on
frozens, using a saponin
			permeabilization of cell membranes.  A couple of
labs have used acetone
			fixed frozen sections, one with a saponin
method, another with TRIS buffered
			saline containing Tween 20.   

			To date, Pharmigen has done only
immunocytochemistry on cells stimulated
			to produce cytokines, and have a very nice
protocol, BUT tissue sections
			have not been worked out by them, in other
words, you will be on your own
			attempting to make the intracellular
immunolocalization of IL-6 work.

			I strongly recommend using monoclonals, and very
good secondaries, some
			labs have used F(ab')2 secondaries, absorbed to
mouse.  IF you succeed
			with your cytokine IHC, there are people waiting
in the wings to know
			what you have done. Be sure you use DAB,
enhanced is nice to have the
			most sensitivy.

			I do not know anyone using paraffin on the IL's
for mouse, I think there
			has been some success with TNF on NBF/paraffin,
but frozens seem to be norm.

			Have been working on cytokine staining steadily
for some
			months and still have poor success, will
continue to tweak until the cows
			come home and the cytokines stain!

			Good luck, you will truly have your work cut out
for you.

			Gayle Callis