GFP staining

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From:Emma Carter <> (by way of Marvin Hanna)
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i am trying to detect GFP in tissue, without much success. We have made
viruses, with GFP as our constitutive marker, and i need to try and find
it using immuno.....I realise i could use fluorescence, but it is not
appropriate in this context.

Originally i was using an anti-GFP polyclonal from Clontech, which
worked fine on paraffins (at a pinch) but not on frozens. However,
Clontech stopped doing this antibody, and replaced it with a peptide
antibody which didnt work.

I found a replacement polyclonal, from molecular probes and am trying to
detect it in frozen material,  but i am having trouble optimising it.

Does anyone have _any_ tips, protocols or advice, before i start to

Current protocol involves fixing in acetone, blocking with H2O2,
blocking with 5% serum, then primary for 1 hour, at varying dilutions,
just to try and get one to work...(NB this tissue is +ve by
fluorescence), washes, then secondary for 1 hour, with 5% serum (i have
used a biotinylated secondary, in which case it is from the Vectastain
kits, and follows their protocol wrt to strepAB, as well as an HRP
conjugated secondary), washed and developed using DAB.

Someone help me!!!!!!!!!!!!!!!!!!!!!!!please!!!!

thanks in advance.

emma carter.

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