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 <[email protected]> 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. > _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
