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
