GFP fixation

tburglin · October 17, 2006, 2:24pm

Originally posted on WormAtlas, by “gidi”, on January 27, 2005

Hi all,
I am having real hard times with fixating embryos/larva for GFP antibody staining. I have tried the usual Methanol/Acetone and the Finney protocols. All worked fine for MH27 controls, but no GFP was detected. Regarding Finney, I used 1-2% PFA and 30’ to 1h30’ defrosting of the animals.
Does someone have a wise advice?
Thanks

tburglin · October 17, 2006, 2:25pm

Originally posted on WormAtlas by “zeynep a”, on February 13, 2005

Try freeze-crack and staining method by Janet Duerr. You can find this and a variation of Ruvkun-Finney method in wormatlas (http://www.wormatlas.org/anatmeth/anatmeth.htm).

tburglin · October 17, 2006, 2:26pm

Originally posted on WormAtlas by “annemckn1”, on July 26, 2005

Duerr’s protocol for freeze cracking says you can use either formaldehyde fixation or methanol/acetone fixation. Is one or the other better for use with GFP antibodies? Also, which GFP antibodies work well for staining? I’ve tried two so far with disappointing results.

Janet_Duerr · November 23, 2006, 12:34am

I realize this is an ancient original post, but someone may still be interested in anti-GFP antibodies. For methanol-acetone fixed worms, I have had good luck with Molecular Probes (now Invitrogen) rabbit anti-GFP and Clontech Living Colors A.V. Peptide Antibody to GFP (#632377, made in rabbit). For formaldehyde fixed tissue, Molecular Probes/Invitrogen monoclonal 3E6 mouse anti-GFP and Chemicon (now Millipore) Chicken anti-GFP (AB16901) have worked well for me.

If you do the fixation and antibody incubations rapidly (within a day), then you may be able to see the endogenous GFP even in methanol+acetone or formaldehyde fixed worms. So, be sure to use a green secondary for the anti-GFP, so any remaining endogenous GFP is the same color as the anti-GFP staining.

operon · December 2, 2006, 12:39am

Hi all,
i am a undergraded student and very much interested in c elegans.Also, I am working on the c elegans project currently. I hope you dont mind to tell me why one still need anti-GFP if the GFP itself is an indicator? we always use GFP fusion protein to study the certain protein’s localization, dont we? why do we need to use anti-GFP for staining? the other question is about the endogenous GFP. Do you mean the worm itself can show gfp?
thank you very much for your advance!

Rookie

Janet Duerr post:4:

I realize this is an ancient original post, but someone may still be interested in anti-GFP antibodies. For [move[/move]methanol-acetone fixed worms, I have had good luck with Molecular Probes (now Invitrogen) rabbit anti-GFP and Clontech Living Colors A.V. Peptide Antibody to GFP (#632377, made in rabbit). For formaldehyde fixed tissue, Molecular Probes/Invitrogen monoclonal 3E6 mouse anti-GFP and Chemicon (now Millipore) Chicken anti-GFP (AB16901) have worked well for me.

If you do the fixation and antibody incubations rapidly (within a day), then you may be able to see the endogenous GFP even in methanol+acetone or formaldehyde fixed worms. So, be sure to use a green secondary for the anti-GFP, so any remaining endogenous GFP is the same color as the anti-GFP staining.

terry_k · January 1, 2007, 4:15pm

I think some fixing protocols can have negative effects on the ability of the GFP to fluoresce. I am not sure what the reason for this is although I guess a good chemist (or protein chemist) would be able to explain it.

My experience with paraformaldehye is that the longer you fix the worms (or cells in tissue culture) the weaker the GFP signal to the point that it can be difficult to detect. I have no experience with the other fixing methods. I decided to try to (i) reduce the amount of time I fixed the samples or (ii) use an anti-GFP antibody. In the end, I was happier with the second option. The antibody for GFP that worked well for me was the rabbit polyclonal anti-GFP antibody from Abcam, ab290. I heard (although I have not personally tried) that their mouse monoclonal antibody is also good.