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 Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet