[Neuroscience] doublecortin immunohistochemistry

wistar_rat me at home.com
Sun Jul 9 19:13:05 EST 2006


We're doing some immunohistochemistry on perfused paraformaldehyde fixed rat 
brain tissue for doublecortin and we're getting a good signal in the dentate 
gyrus but there's lots of speckled non-specific labelling in the cell 
layers.  We've tried various dilutions of blocking serum to get rid of it. 
It takes care of the diffuse background but not these specks.  They don't 
occur when only the fluorescent secondary is used so I think it's the 
primary that's binding to other things.  The papers I've read show fantastic 
labelling using procedures that sound identical to ours.  I don't see this 
with other fluorescent labelling in this tissue (e.g. NeuN, BrdU, 
calbindin).

We've tried it on old tissue stored in sucrose/ethylene glycol as well as on 
freshly cut (cryostat) tissue put directly into saline but the results are 
the same.  Can anyone suggest anything that will help?

The primary is goat-anti-DCX and the secondary is donkey-anti-goat-alexa.

Thanks.

Brian




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