Re: Fixing GFP positive tissue after freeze sectioning

Posted by Melissa Gonzalez on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Far-red-filter-set-tp592652p592660.html

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Can you directly observe GFP in fresh frozen tissues?
I always fix in 4% PFA, cryoprotect, and then freeze. Those sections
always look great.

But any time I have received fresh frozens they turn up negative. Post
fixing in PFA doesn't work either.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Caroline Bass
Sent: Monday, March 10, 2008 6:55 AM
To: [hidden email]
Subject: Re: Fixing GFP positive tissue after freeze sectioning

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I regularly fix brain tissue that is expressing GFP (via injection of a
viral vector).  Standard NBF works fine and I have no problems.  I
looked
into this issue extensively a few years ago, and I can say that two
things
killed my signal: not fixing the tissue, and allowing fresh tissue to
dehydrate.

I have fixed my brains in two ways, perfusion of the animal and
immersion in
NBF.  Since the brain (mouse) is a large organ I cut it in three parts
and
just dropped it in for a quick and easy fix.  This worked fine, but I
only
used it when I needed to get a quick look at the expression zone.


On 3/10/08 9:34 AM, "Kirill Ukhanov" <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Hege,
>
> GFP could be quenched by aldehyde fixation, on the other hand you need
to
> fix the tissue to avoid wash out of GFP as it is mostly located in
cytoplasm
> (well, depending how its expression is controlled). Poor fixation may
result
> in a loss of the GFP from the tissue even if you think of not using
> permeabilization etc. I would recommend fixing your tissue before
> cryosectioning and then use anti-GFP antibody to enhance native GFP
(if you
> find the signal too weak). I remember I found an extensive discussion
on

> this issue here in the list or elsewhere in microscopy forums.
>
> good luck
>
> Kirill Ukhanov
> University of Florida
>
>
> On Fri, 7 Mar 2008 16:22:06 +0100, Hege Avsnes Dale
> <[hidden email]> wrote:
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hi.
>> Do anyone have a protocol for good preservation of GFP in tissue

after
>> freeze sectioning?
>> The tissue is a tumor with EGFP-expressing cells and need only a
>> fixation that will preserve GFP (no permabilization  needed). Would
it
>> also be a better idea to fix before sectioning?
>>
>> Hege
>>
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