The objective of this core TR&D project is to improye EM methods for in situ hybridization. We have achieved exciting progress in localizing nucleic acid sequences at the electron microscopic level. A project using a pre-embedding approach to localize Poly (A)+ mRNA in hippocampal neurons has been completed and was presented at this year's Society for Neurosciences meeting. This study was the first anatomical demonstration that mRNA is found at the base of dendritic spines and in the vicinity of post-synaptic densities. A manuscript detailing this work is in preparation. We have also continued efforts to localize individual messages using this approach and the photo-oxidation approach detailed in last year's report. We are still encountering sensitivity problems at the level of detecting single messages. We are currendy exploring amplification methods based on the tyramide system developed by Dupont. Tyramide is a substrate of peroxidase and when activated by this enzyme, it sticks to sites near the site of activation. Tyramide can be conjugated to biotin or to a fluorophore and thus can be used for signal amplification. In our initial studies with immuno detection, the tyramide system yielded a tremendous increase in signal detection. We are planning to use this methodology on the in situ hybridization work to determine whether sufficient signal amplification occurs to detect individual messages in the nucleus or cytoplasm.
NIH ExPorter
NIH ExPorter