Re: problem with fresh frozen eGFP sample

Posted by Jean Brennan on
URL: http://confocal-microscopy-list.275.s1.nabble.com/problem-with-fresh-frozen-eGFP-sample-tp593098p593111.html

Hi Ian,

 

I do see that now.  I will try some tissue in liquid Nitrogen and some in the vapor of liquid nitrogen to see if I can avoid water crystals.  It becomes interesting when I need the fresh tissue for RNA.

Best,

Jean

 

Jean Brennan
Research Specialist
Rothstein lab, Dept of Neurology
Johns Hopkins University
600 N. Wolfe St., Meyer 6-174
Baltimore, MD  21287
410-614-4119; FAX: [phone]
[hidden email]

>>> Ian Dobbie <[hidden email]> 04/14/08 6:40 AM >>>

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Jean Brennan <[hidden email]> writes:

> Hi all,
> 
> I have an animal that is Tg eGFP for a neurotransmitter in astrocytes.  It
> shows up very well in brain and spinal cord after perfusing with 4%PFA.  I'm
> trying to Laser capture some cells for RNA work but having trouble visualizing
> the cells in the fresh frozen tissue.  I'm doing cervical dislocation, the
> disection is under 1min., then placing the tissue into 2-Methylbuane that has
> been cooled and surrounded by dry ice.  The tissue is then cut in a cryostat,

GFP fluorescence is completely dependant upon structural water within
the beta barrel[1]. I'm not sure how strongly you are dehydrating your
sample, but this could cause you to loose you GFP signal.

Ian

[1] see Tsien, RY. THE GREEN FLUORESCENT PROTEIN
Annu. Rev. Biochem. 1998.67:509-544.