I am staning formalin-fixed paraffin embedded mouse brain with Fluoro Jade C and DAPI. These brains are from animals that had a global ischemic injury. I am getting beautiful bright fluorescence with the FJ but also some cells that are faintly fluorescent with FJ and with DAPI appear slightly shrunken and misshapen and usually have a primary process labelled. To my eye it looks very similar to the 'dark neuron' phenomenon that is often discussed with conventional H&E or Nissl techniques. I am trying to count these labelled cells and my concern is that these cells have been dead longer and thus are no longer taking up the FJ and so if I don't count them I may dramatically underestimate my injury. On the other hand I don't want to count artefactual labelling. Does anyone have a suggestion? I can send pictures if that will help.
thanks very much,
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