[Histonet] Re: Fixation for EM

[Chana de Wolf]

By the way, I should have mentioned that these are RAT brains I am
talking about.

Sorry!


On Tue, May 5, 2009 at 3:50 PM, Chana de Wolf <chana.de.wolf <@t> gmail.com> wrote:
> Hello all, I am new to Histonet and very happy to be here! Of course,
> I was brought here the same way most are -- I have questions!
>
> I am currently working on a project for a mathematician who wants to
> develop an algorithm that can be used to calculate length of ischemia
> when fed EM images of ischemic brains. In order to develop the
> algorithm, we must first generate EM images of brains after various
> periods of ischemia. Like most histologists, any brain fixations I
> have done have involved immediate perfusion fixation to *minimize*
> ischemia, so this is new and interesting territory.
>
> Based on my own experiences and a thorough reading of the literature,
> I assume that perfusion fixation of ischemic brains is not practical
> due to the "no-reflow" phenomenon. My proposal, then, is to hemisect
> and diffusion fix the brains. However, *I* am not performing the EMs,
> and I am not totally certain when the EMs will take place following
> fixation.
>
> My questions follow:
>
> (1) What is the best fixative solution to be used under the
> circumstances? I was thinking of using a combination of
> paraformaldehyde and acrolein due to acrolein's penetrative qualities
> and superior fixative strength.
>
> (2) Do I need to perform a secondary fix with osmium tetroxide
> myself...or do I leave that to the EM lab techs to perform when they
> make slices? If at all possible, I want to minimize the amount of
> tissue processing on my end.
>
> (3) How long can the brains be left in post-fixative solution prior to
> further processing in the EM lab? Or, in other words, is there a
> maximum time period after which fixed whole brains cannot be sliced,
> washed, embedded, or otherwise processed?
>
> (4) Any other suggestions, comments, etc.? Obviously, there has to be
> a less-than-ideal division of labor in this matter because we do not
> expect the EM lab to remove brains from rats that have been dead for
> up to 48 hours. I can handle that part, but I want to ensure I do
> everything right because what I do affects the rest of this study.
>
> Thank you for your time and any valuable insights into this matter.
>
> Chana de Wolf
> Advanced Neural Biosciences, Inc.
>