[Histonet] quick and easy sectioning of fresh brain

Caroline Bass cbass <@t> bidmc.harvard.edu
Tue Aug 14 23:46:41 CDT 2007

Hey Folks,

So I want to have my cake and eat it too.  I have injected a rat with  
a virus that will express EGFP.  Ideally I'd like to collect the  
fresh brain, cut off thick floating sections, enough to get a feel  
for the spread of the virus, and possible punch out the EGFP area to  
get mRNA and/or protein for qPCR and western blot.  Are there any  
suggestions for doing this?  I have access to cryostats, sliding  
microtomes, and brain molds.  Cutting fresh brain is difficult, so  
I'd like to freeze it to section on the sliding microtome, collect a  
floating section, and visualize under a fluorescent dissecting  
microscope.  Are there any issues with doing this?  How should I  
freeze the brain, and how will this affect mRNA and protein levels?   
How difficult are unfixed floating sections to deal with?  Finally,  
can I take some sections and fix by immersion in PFA so that I can  
store for longer periods of time.

Any suggestions would be appreciated.  I've had very good luck  
working with perfused/fixed tissue, and would like to continue with  
it, but it doesn't seem possible with the mRNA/protein endpoints.



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