Hi,
I'm currently trying to detect TNF via IF in mouse leukocytes (specifically neutrophils but will take what I can get at the moment).
I have tried a range of positive controls (splenocytes activated with PMA and BFA for 3h; peritoneal cells collected post acute peritonitis) and a range of different commercially-available antibodies, yet I am struggling to find a signal that is above the level of the isotype control.
Does anybody have any experience with detecting TNF in this way? Does anybody know of any good antibodies against it? Does anybody have any better ideas wrt. positive controls?
Any help would be valuable at this stage.
Cheers
Ross
Just as theory, all of your cells investigated, have Fc receptors, i think. Might be your isotype control bind too much?
Fc receptor binding is not really a problem. When you have a good anti-cytokine antibody it works well and if you are not getting background membrane staining of macrophages at sensible antibody concentrations - which you should not - the do not worry about FcRs. The problem is finding a good IF antibody. We tried to pin down good antiTNF antibodies for immunostaining 25 years ago and gave up. Immunoperoxidase or alkaline phosphatase was more use for the cytokines we did get believable signals for (e.g. gamma IFN). I would have thought that in the intervening time someone would have got TNF antibodies sorted but I wouldn't bet on it. I am afraid I have lost touch with who is getting good cytokine pictures these days.
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