Victor and Jacqui,
Could "some" neurons appearing to be immunoreactive to TNF alpha or Il-1 beta simply be the fact that they are cytokines? Small molecular weight, they are meant to be released from cells and not membrane bound there or as part of the cell structure. Glial cells, by the very fact of what they are, are intimately associated with neurons. How was tissue fixed? Maybe they are dispersed or leaking to a neighboring neuron. Many targets are anchored where they are, such as your NeuN label, and that target is not going anywhere. Cytokines do. The references of 10-15 years ago were novel then. Not so now. TNK K/o as you said you can get. At Wash State, you should have a good mouse transgenics and mouse k/o husbandry facility. With some Balb/c's, they should be able to generate IL-1 alpha -/-. beta -/- or alpha/beta double knock/outs. Not that difficult for a good knock out genetics lab. If you are using the knock outs as control for the IHC staining (and this applies to any use
of knock-outs as negative controls for any IHC staining) be sure you know EXACTLY what is knocked out. A mouse frizzle/frazzle knock-out does not imply the total absence of mouse frizzle/frazzel gene and protein. One particular coding exon in a multi-exon gene could leave the protein unresponsive or unusable in-vivo or shortened but enough might be left, and if the proper epitope is left, for IHC staining to occur. Knock out is not the same as complete absence. So you can stain something that is "knocked out" and that is why you need to be aware of EXACTLY what is knocked out. Many people, including yours truely, has been caught like this.
-------------- Original message --------------
From: "Jacqui Detmar"
> Hi Victor.
> IL-1alpha was knocked out by Iwakura's group in 1998 (see Horai et al.,
> 1998). IL-1beta was knocked out by David Chaplin's group (see Shornick
> et al., 1996) and Lex Van der Ploeg's group (see Zheng et al., 1995).
> Have you tried depleting the antibody with the peptide immunogen? Is
> this even possible for these antibodies? Just a thought. Good luck
> Jacqui Detmar, Post-doctoral Fellow
> Samuel Lunenfeld Research Institute, room 876
> Mount Sinai Hospital
> 600 University Avenue
> Toronto, ON, Canada
> M5G 1X5
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