The objective of this core TR&D project is to improve EMmethods for in situ hybridization. We have achieved exciting progress in localizing nucleic acid sequences at the electron microscopic level. We are still encountering sensitivity problems at the level of detecting single messages. We are currently 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 have used this methodology routinely now in non-radioactive light microscopic detection of K+ channel transcripts in cultured neurons and tissue sections and are planning on extending this analysis to the electron microscopic level in the near future. We are also beginning to work with riboprobes instead of the cDNA and oligoprobes that we have used previously. Riboprobes should afford improved sensitivity although their use necessitates more stringent wash conditions which severely impact ultrastructure. We are considering the use of post-embedding methods as well as more robust fixation protocols to overcome expected morphological damage.
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