Abstract
In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. We describe our EM-ISH method using biotinylated oligonucleotide probes for rat growth hormone (GH) and prolactin (PRL) mRNAs and compare the preembedding method with the postembedding method. The hybridization signal intensity by the postembedding method is lower, and non-specific signals are relatively frequent, in comparison with the preembedding method. The preembedding method thus appears to be easier and better than the postembedding method from the viewpoint of applicability and preservation of mRNA. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. Modulations of ultrastructural mRNA expression induced by stimulatory or inhibitory factors can also be detected with EM-ISH method. The simultaneous visualization of mRNA and encoded protein in the same cells using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex is also described. This ultrastructural double staining method for mRNA and encoded protein can be expected to provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein.
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