anti-GFP antibody staining

I am having issues troubleshooting an anti-GFP antibody staining. I have a transgenic strain harboring a gfp construct, that when fixed, exhibits a decrease in GFP intensity, so I am attempting to do a double staining with anti-gfp and anti-mec-18. Before I waste my valuable mec-18 antibody, I want to make sure I can do a staining with anti-gfp. I am following the Gary Ruvkun and Michael Finney protocol on wormatlas, but I am not able to detect GFP. I am using anti-green fluorescent protein, mouse IgG2a as my primary and Alexa Fluor 488 goat anti-mouse IgG, IgM(H+L) as my secondary. Both were recently purchased from invitrogen. I have incubated with the primary antibody at concentrations ranging from 1:50 to 1:200 and the secondary at concentrations ranging from 1:100 to 1:1000. In any of these cases, my staining did not work. Does anyone have any suggestions I can try to troubleshoot, or has anyone been successful at staining with an anti-gfp antibody? If so, what protocol and antibodies were used?

I would really appreciate any input/adviceā€¦


I have not used the Invitrogen anti-GFP antibody, but it is possible that the fixation and post-fixation treatment in the Ruvkun-Finney protocol is disrupting the antibody binding site for that particular antibody, especially if it is a monoclonal. It is also possible that you have not gotten good permeability with the post-fix treatment - are you sure that your animals are permeable to antibodies in general?

I have had luck with Chicken anti-GFP from Chemicon (now Millipore) on formaldehyde fixed worms. So if you are sure that your worms are permeable, you might want to try this antibody.

Good Luck, Janet