Intracellular staining of cultured macrophages
Nasos Dagkalis
Monday, 16 March 2009 07:53 UTC
Hello everyone,
I’m trying to stain cultured mouse macrophages intracellularly for Arginase, iNOS and other markers but keep hitting snags.
I should mention that I’m trying to block Fc receptors with a cocktail of rat, rabbit and goat sera (5-10%) prior to staining, as my secondary antibodies are raised in these hosts. That usually works fine.
I use Saponin buffer for permeabilisation. The other problem is that I have to use secondary antibodies, which will probably increase the background. Any opinions on how to circumvent that? I’m at a loss as to what will allow me to tell a real signal from background. My main concern is how many and what blocking steps should I use.
Many thanks
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Replies
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Hey Nasos,
If you don’t want to use secondary antibodies, try labelling your primaries directly. These kits have worked well for me:http://www.piercenet.com/products/browse.cfm?fldID=294EFD98-4461-4352-A046-50B522F52952.
If not you can always biotin label the one primary that gives the most background and detect with streptavidin or one of the more specific avidin conjugates.
Hope this helps,
Tarl
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