[Enzyme-immuno-histo in situ hybridization]

In order to understand the physiological functions of normal as well as neoplastic cells, it is best to investigate at the level of individual cell. We have been involved for the past several years with the development of a method for the localization of mRNA in individual cells using hapten labeled cDNA. Specifically, cDNA are labeled with dinitrophenyl (DNP) and the labeled cDNA are hybridized with mRNA in cells. Then the hybridized DNP-cDNA are then localized immunohistochemically using peroxidase-labeled antibodies. In this investigation, we concentrated on establishing the best condition for the removal of proteins in order to expose mRNA and for the removal of non-specific back ground staining. It was found that some proteins treatment was necessary for the exposure of mRNA for well fixed tissues and that high concentration of formamide and low concentration of salt removed effectively the nonspecific staining. Using the backscattered electron imaging for by scanning electron microscope, specific mRNA was localized in cells and tissues at the ultrastructural level.