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immunostaining but GFP is bleached - (Oct/16/2006 )

I am staining some protein molecules in C. elegans body wall muscle. I aslo want to check the GFP after immunostaining.( It is a GFP fusion protein rescue line. ) Unfortunately the GFP is bleached. I have tried to incubate the 1st Antibody 1 hour, 2hours or overnight. It doesn't help a lot if I shorten the incubation time, actually it makes the staining worse. I fix the embryo in 4%PFA. I am thinking to do GFP and my protein of insterest double staining if I can't solve the GFP bleaching problem. It is important for me to check my GFP fusion protein and other protein molecules co-localization.

Can anybody give me some suggetions on this problem? Thank you very much.



have u looked at the GFP flurescence with out the immunostaining. I mean if u fixed the tissue, mounted it and could u observe good fluorescence with GFP.

try using mounting media which prevents bleaching as well.