Thanks for the nod Gayle. We've converted to Rockland goat anti-GFP after having some reproducibility issues with the rabbit polyclonal we were using from Novus.
I agree with your recommendation to use an indirect IHC method to increase the signal. If she's using a rabbit anti-GFP/Alexa 488 she can still come back with an anti-rabbit Alexa 488 and see if that increases the signal that way. She might also try a biotinylated anti-rabbit, and come back with a Streptavidin Alexa 488. I so do not like FITC, have watched it photobleach as I was viewing it.
I will leave it to my IHC Specialist to give the details of her experience with using the Rockland goat antibody. I think it works equally well using antigen retrieval or Proteinase K on formalin fixed paraffin embedded animal tissues. And she's used it both with the goat polymer kit and regular anti-goat Alexa 488.
Sharon, care to elaborate on the details of your staining protocols?
Teri
-----Original Message-----
From: Gayle Callis [mailto:gayle.callis@bresnan.net]
Sent: Tuesday, November 18, 2008 11:04 AM
To: MaryAnn Dixon; histonet@lists.utsouthwestern.edu
Cc: Johnson, Teri
Subject: Re: [Histonet] anti-GFP
The problem may be that you are using a directly conjugated antiGFP rather than a rabbit antiGFP followed by coming back with a secondary conjugated to Alea Fluor 488. This is called quenching, a phenomenom, where fluorophore moleculas too close together on cells or in tissues tend to cancel out the fluorecsing ability of the fluorophore. You should go onto internet and look at a fluorescence Jablonski diagram which show how this occurs, Olympus website also wonderful discussions in pdf form, for all fluorescence applications, including this diagram - for confocal and fluorescent microscopes.
I suggest you stain for GFP (Teri Johnson method) where you retrieve, use a rabbit antiGFP, then come back with a secondary either conjugated to FITC (Jackson has excellent antibodies, or one of the Cy fluorophores, or better
yet, Goat antirabbit-Alexa 488. Be sure you use Molecular Probes Prolong
gold antifade mounting media after staining - this is superior for preventing fading of fluorophores, even 488. Not all aqueous mounting medias will prevent fading of fluorophores, even the Alexa dyes.
Teri recommends rabbit antiGFP rather than Goat antiGFP for paraffin work, as the rabbit hosted antibody gave less background than the goat antiGFP.
One can also purchase Rabbit antiGFP from Rockland.
I made a CC to Teri Johnson so she is in this email loop. You may want to discuss this problem with her, and what antigen recovery method she prefers.
If worse comes to worse, and you can't afford another antibody, use the antiGFP-488, come back with an antiAlexa 488 (Molecular Probes) and detect that antibody with an antibody that has FITC, or the appropriate fluorophore. A round about way, but the same type of technic used to detect FITC.
Gayle M. Callis
HTL(ASCP)HT,MT
----- Original Message -----
From: "MaryAnn Dixon"
To:
Sent: Tuesday, November 18, 2008 9:19 AM
Subject: [Histonet] anti-GFP
Hi histonetters,
I stumbled into immunofluorescence for the first time and could use some advice. I am trying to stain GFP on formalin fixed paraffin embedded sections. I have a conjugated alexa fluor 488 anti-gfp antibody from invitrogen that I've now found out was not tested on paraffin sections. I have seen articles supporting and denying that it works. In addition, do I retrieve or not as again, I've seen literature supporting both. Moreover, one article cut sections at 12 microns. My protocol for my first run consisted of a protein block for 10 minutes, blowing off, 1:400 of the conjugated alexa fluor 488 ant-gfp antibody for 1 hour at room temp., buffer rinse, DI water rinse, aqueous mounting medium, and coverslip. To my best ability I performed everything in the dark. The results were that I had no fluorescing whatsoever!! Any help would be appreciated.
MaryAnn Dixon BS
Biological Scientist
Anatomic Pathology
UF Veterinary Medical Center
(352) 392-2235 Ext. 4517
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