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
antibodies.
I would like to see Sharon's protocol too.
Gayle M. Callis
HTL(ASCP)HT,MT
----- Original Message -----
From: "Johnson, Teri" <[EMAIL PROTECTED]>
To: "'Gayle Callis'" <[EMAIL PROTECTED]>; "MaryAnn Dixon"
<[EMAIL PROTECTED]>; <histonet@lists.utsouthwestern.edu>
Cc: "Beckham, Sharon" <[EMAIL PROTECTED]>
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?
Teri
-----Original Message-----
From: Gayle Callis [mailto:[EMAIL PROTECTED]
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" <[EMAIL PROTECTED]>
To: <histonet@lists.utsouthwestern.edu>
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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