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Jean Brennan <[log in to unmask]> 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.