Hi everybody,

I had a question regarding fixing rat brain. We are currently  
perfusing and fixing with a 4% para / 3% sucrose solution. I am trying  
to get X-Gal staining, but I have read this may be undetectable once  
tissue has been paraffin embedded.  First off, has anybody had luck  
with paraffinized tissue and x-gal? Secondly, we wanted to snap freeze  
and section the tissue ourselves if paraffin was not an option.  From  
what I've tried thus far, I am not getting the structural integrity  
that comes from paraffinzed tissue.  I know you are never going to get  
as good slides when frozen, but I think it should look more intact  
than what I am seeing.  The tissue looks stretched out and very gappy.  
Could one of the reasons be that the tissue is being overfixed? The  
tissue has been sitting in the para/sucrose sol'n for anywhere from 3  
days to 4 weeks. Maybe my snap freezing technique is off, but I don't  
think ice crystallization is the problem. Is there any advantage to  
using the para/sucrose sol'n to fix, or is it just better to fix  
overnight, move to sucrose gradient and then snap freeze?

Thanks in advance for suggestions.

-Eric

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