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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