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.
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