RE: [Histonet] brain fixation and native EGFP fluorescence

From:"Charles Scouten"

See the following link for a through discussion of perfusion, and how to
avoid soft tissue shrinkage.

Sucrose should be used as the prewash, not in the fixative, at least
until the tissue is mostly fixed.

Charles W.  Scouten, Ph.D. 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300 x 342
FAX  314 522 0377 

-----Original Message-----
[] On Behalf Of Caroline
Sent: Tuesday, January 03, 2006 9:25 PM
Subject: [Histonet] brain fixation and native EGFP fluorescence

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?


Caroline Bass

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