Electron microscopic observation of intracellular expression of mRNA and its protein product: Technical review on ultrastructural in situ hybridization and its combination with immunohistochemistry

In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. Three different approaches have been applied by the investigators in this EM-ISH study: preembedding method; non-embedding method using ultrathin frozen sections; and postembedding method. In order to obtain satisfactory morphological preservation and retain the messages, we routinely utilized 6 pmthick frozen sections fixed in 4% paraformaldehyde for the preembedding method and tissues embedded in LR White resin for the postembedding method. The hybridization signal intensity by the postembedding method was lower, and non-specific signals were 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, although quantitative analysis of the expression of mRNA is rather difficult in the preembedding method. 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. 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