Re: [Histonet] brain fixation and native EGFP fluorescence

From:John Kiernan

A colleague who knows more than I about these
matters tells me that the native fluorescence of
GFP depends critically on the highly organized
barrel-shaped configuration of the molecule -
something that may be preserved or destroyed by
fixation, but won't survive dehydration in
alcohols etc. Remember that aldehyde-fixed and
even unfixed
 tissues can have green autofluorescence when
exited by blue light.

Heavy metal salts generally tend to suppress
fluorescence, even though some histochemical
fluorescence methods can be enhanced by zinc or
aluminium salts. It might be best to avoid zinc
formaldehyde mixtures for this reason, and also
because zinc ions coagulate proteins and can be
expected to change the shape of the green
fluorescent protein molecule, perhaps making it no
longer fluorescent.

John Kiernan
Anatomy, UWO
London, Canada
Caroline Bass wrote:
> Hello Everyone,
> I have been using a viral vector to express EGFP in the mouse liver
> and examining native fluorescence.  I generally fix the tissue by
> cutting the liver into blocks, immersing in NBF and sectioning on a
> cryostat.  I have gotten some really good results just looking at
> native fluorescence.  I am hoping to extend this to the brain.  I
> have now injected my viral vector into the striatum of a mouse and am
> set to collect the tissue.  Since I am working with the brain, my
> natural tendency is to perfuse, as I have always heard this is the
> best method of fixing brain tissue.
> Does anyone have advice in terms of picking a good fixative for
> perfusion?  I have been reading about a lot of different fixatives
> for the brain, one which includes sucrose.  I am familiar with
> sucrose cryopreservation, but I haven't considered adding it to the fix.
> Any suggestions would be appreciated.  If you have a suggestion
> please include the formula.  Someone recently suggested a
> paraformaldehyde zinc fix to me.  What are the advantages of this
> paraformaldehyde alone and does anyone have a formulation for it?
> Thanks,
> Caroline Bass
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