Re: [Histonet] anti-GFP

From:"Gayle Callis"

Thanks Teri,

We prefer to use Goat antiGFP-biotinylated.  This allows us to come back 
with Molecular Probes Streptavidin-Alexa 488 and eliminates any secondary. 
Be careful though, endogenous biotin can bite you on this one.    One has to 
use an avidin/biotin block, and we now use Vectors Streptavidin-biotin 
block, as this has some special considerations that SA binds to some 
addressins and epithelial cells.  This is in the literature.

Our protocol is simple, since we do not work with NBF or PFA fixed tissue- 
only fresh snap frozen tissue sections,  and avoid any antigen retrieval. 
Our solvent fixation ruins GFP  fluorescence which means immunostaining for 
the GFP.  We rarely use antiGFP to detect just GFP, but always as a double 
IFA stain with a murine CD marker or some other marker.

Rockland is an excellent source for antibodies against GFP but also for Red 
Fluorescent Protein (DsRed from Discosoma sp. - hope I spelled that 
correctly).  This can be confusing since one of the chimeras of GFP is also 
called RFP, from Aqueora jellyfish.  Be careful when shopping for these 

I would like to see Sharon's protocol too.

Gayle M. Callis

----- Original Message ----- 
From: "Johnson, Teri" 
To: "'Gayle Callis'" ; "MaryAnn Dixon" 
Cc: "Beckham, Sharon" 
Sent: Tuesday, November 18, 2008 10:24 AM
Subject: RE: [Histonet] anti-GFP

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?


-----Original Message-----
From: Gayle Callis []
Sent: Tuesday, November 18, 2008 11:04 AM
To: MaryAnn Dixon;
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 

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 

Gayle M. Callis

----- Original Message -----
From: "MaryAnn Dixon" 
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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