Using DAF-FM (NOS marker) in embedded sections - (Apr/04/2007 )
Has anyone used DAF-FM (invitrogen), which is a fluorescent marker for nitrogen oxide, with mouse tissue embedded in OCT?
The only papers i can find that use DAF-FM are with living cells.
Do you mean NOS (nitric oxide synthase) or NO (nitric oxide)?. If you mean NOS, you cannot use DAF-FM to stain for NOS (the enzyme family that catalyes NO formation) in fixed cells. DAF-FM is cell-permeable, and reacts with NO free radicals, becoming insoluble and fluorescent in the presence of NO. NO is very labile and reactive so you have to have a active generation of NO to get a detectable DAF-FM precipitation and fluorescence. Note: Freezing will not fix NO. See this site http://probes.invitrogen.com/media/pis/mp23841.pdf
If, however, you want to stain for NOS in fixed cells , use an antibody for the enzyme of interest (eg iNOS, eNOS, or nNOS).
Also, while NO is too labile and reactive to demonstrate in fixed tissues, you can stain for evidence of its former reactivity. NO reacts with proteins in a pretty well understood mechanism, specifically nitorsylation of the 3`OH of tyrosine residues. You can use an antibody to stain for this modification. I cant think of a specific Ab right now, but if you google 3-nitro-tyrosine, you should find several ab's.
Thanks for the help. I will be using an antibody to iNOS now.