Re: GFP tissue preparation for confocal microscopy

Posted by Eric Scarfone on
URL: http://confocal-microscopy-list.275.s1.nabble.com/GFP-tissue-preparation-for-confocal-microscopy-tp2680696p2684164.html

I agree with Stephen. For all purposes, PFA should really be prepared "extemporaneously", but I find it difficult to convince student to do that these days.....
Nowadays with aliquot and freeze PFA (we use small amounts on cell cultures). Not problem with GFP so far! 

Some detection problems may be linked to the amount of expression one gets. At low level the signal will be lost in the background fluorescence (green) induced by aldehydes. In this case countestaining works well.
Another problem maybe bad or slow fixation. If your construct is cytoplasmic it will leak out of cells....

Cheers

Eric

Eric Scarfone, PhD, CNRS,
Center for Hearing and communication Research
Department of Clinical Neuroscience
Karolinska Institutet

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----- Original Message -----
From: Stephen Bunnell <[hidden email]>
Date: Thursday, April 23, 2009 5:20 pm
Subject: Re: GFP tissue preparation for confocal microscopy
To: [hidden email]

> PFA does not kill GFP fluorescence.
>
> BAD PFA kills GFP fluorescence.
>
> We routinely use 1% PFA with GFP, YFP, and CFP, and have never had
> a problem
> so long as the PFA is fresh and pH¹ed to between 7.0 and 7.4.
>
> -Steve
>
>
>

> On 4/22/09 10:34 PM, "Young Jik Kwon" <[hidden email]> wrote:

>
> > All,
> >
> > We are trying to take confocal micrographs of at tumor tissues
> that express
> > GFP. What would be the sample preparation procedures? We tried
> cryosectioned> samples but the cell morphology seems weird. We
> already know paraformaldehyde
> > fixation kills GFP fluorescence. Any expert's advice?
> >
> > Best,
> >
> > Young
> >
> >
> >
>
>
> ****************************************************************************
> Stephen C. Bunnell, Ph.D.
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> Tufts University Medical School
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