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 essen tial for elucidating the intracellular distribution and role of mRNA in pro tein synthesis. Three different approaches have been applied by the investi gators in this EM-ISH study: preembedding method; non-embedding method usin g ultrathin frozen sections; and postembedding method. In order to obtain s atisfactory morphological preservation and retain the messages, we routinel y utilized 6 mu m-thick frozen sections fixed in 4% paraformaldehyde for th e preembedding method and tissues embedded in LR White resin for the postem bedding method. The hybridization signal intensity by the postembedding met hod was lower, and non-specific signals were relatively frequent, in compar ison with the preembedding method. The preembedding method thus appears to be easier and better than the postembedding method from the viewpoint of ap plicability 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 loca lization of mRNA and the exact site of specific hormone synthesis on the ro ugh endoplasmic reticulum. The simultaneous visualization of mRNA and encod ed protein in the same cells using preembedding EM-ISH and subsequent poste mbedding immunoreaction with protein A colloidal gold complex is also descr ibed. This ultrastructural double-staining method for mRNA and encoded prot ein can be expected to provide an important clue for elucidating the intrac ellular correlation of mRNA translation and secretion of translated protein .