Electron Microscopy and In Situ Hybridization
This chapter describes a pre-embedding in situ hybridization method utilizing an immunogold-silver intensification step to identify P2Y2 receptor mRNA transcripts in the adult rat cerebellum. The method was applied for ultrastructural (electron microscopic) examination. Transcripts for P2Y2 receptors, marked by the gold-silver grains, were revealed in Purkinje cells. Transcripts were essentially localized in the cytoplasm although they also appeared to be specifically associated with granular endoplasmic reticulum. This suggests that Purkinje cells may produce functional P2Y2 receptors involved in the ATP-related regulatory role in the cerebellum. The in situ hybridization method that was applied enabled simultaneous preservation of tissue ultrastructure and localization of labeled mRNA transcripts. This method may be adapted and used to study various tissues.
Key WordsIn situ hybridization ultrastructure cerebellum P2Y2 receptor
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