[ExI] Another step towards uploading
rafal.smigrodzki at gmail.com
Fri Oct 11 04:58:16 UTC 2013
On Wed, Oct 9, 2013 at 10:18 AM, John Clark <johnkclark at gmail.com> wrote:
> On Tue, Oct 8, 2013 at 1:11 AM, Rafal Smigrodzki
> <rafal.smigrodzki at gmail.com> wrote:
>> > You need to take into account the damage introduced during mechanical
>> > slicing. Even a single bad slice (torn, crushed, happens all the time, as
>> > anybody who spent hours at a microtome can attest) could scramble the
>> > long-distance fibers
> I'm no expert but just from my experience of using a meat slicer I would
> think mangled slices could be a problem if you were trying to make the
> slices super thin, but I'm talking about slices on the order of a centimeter
> thick or maybe even more, and only about a dozen slices. And if the blade
> was sharp and the gap between the slices narrow it should be possible to
> deduce what those long-distance fibers did in that missing gap.
### This might be an option, especially with brains that cannot be
perfused because they spent too much time in ischemia and there was
blood clotting in the vessels. Such brains might be a lost cause
anyway but who knows, fixation by immersion might here be better than
bulk freezing. Either way, you want to avoid this situation.
> method was used wouldn't slices ensure better distribution of cryoprotective
> or chemical fixative than entire uncut brains?
### Perfusion always gives better distribution than immersion. You
really want to avoid a situation where perfusion, whether cryo or
fixative, is no longer possible. If slicing is your only option, you
might be toast anyway.
Let me put it this way: Either you are perfusable or not. If yes, then
we can argue about the relative merits of cryoperfusion vs. fixative
perfusion, and I will grant this is not an open-and-shut case. If you
are not perfusable, the damage induced by slicing might be the least
of your worries and the whole exercise might be futile, although
fixation by immersion followed by sucrose gradient and cryogenic
storage might be the best of your (dismal) options.
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