[Histonet] anti-GFP

[James Watson]

We use Zamboni's fixative and are able to use paraffin sections.  We do
use 5% glycerin in our 100% denatured alcohol so that might be
protecting the GFP from being damaged during dehydration. 

jwatson

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Emily
Sours
Sent: Tuesday, November 18, 2008 8:47 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] anti-GFP

We've tried Invitrogen's rabbit anti-GFP on paraffin sections and it
doesn't
work with DAB or fluorescent label.
Our fix is Carnoy's or butanol, and we cut at 10 microns.
With cryosectioning, the antibody works perfect, so it's definitely not
the
antibody.
I'm not sure what in the paraffin processing would destroy the GFP
antigen,
but apparently something does.

Emily

On Tue, Nov 18, 2008 at 11:19 AM, MaryAnn Dixon
<DixonM <@t> vetmed.ufl.edu>wrote:

> 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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