Help! GFP is bleached after immunostaining. - (Oct/16/2006 )

Hi,
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.

Qian

some aspects:

was there an initial good GFP expression (to check by life cell imaging)? Do you use original GFP or EGFP and equivalents (they are more resistant against bleaching)? Do you use Dabco in your mounting medium (highly recommended even in your case)? There some other anti-bleaching agents, look at Molecular probes/invitrogen. Incubations are performed in dark, aren´t it?

We have good experience with EXFP + immunostaining; incubation time for antibodies should be no problem; if you have good GFP expression, and care against bleaching, GFP fluorescence is stable for
a good while

The original GFP is OK, I can take nice pictures of it. The problem is that after staining GFP signal is so weak at the moment I mounted on the microscope. So I was thinking it is bleached during antibody staining. It is just my guess. I will try the dabco in mounting buffer. My question is if bleaching really happens during staining, can I add dabco in TPBS to wash or mix with primary antibody to do the incubation? Will that help or will it ruin the staining?Thanks