[Histonet] Fixation for EM

[Chana de Wolf]

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.