Posted by
Caroline Bass on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Far-red-filter-set-tp592652p592659.html
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalI 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
>> =========================================================================