[In situ localization of mRNA with T-T dimerized DNA probes]

Abstract

When a particular antigen is immunohistochemically localized at a given site, it is difficult to differentiate whether the antigen was produced at the site or immigrated to the site from elsewhere. If the antigen, e.g. hormone, is synthesized at the site, it indicates that the site is the effector cell and if the antigen is not synthesized at the site, it indicates that the site is the affector cell. Hence this differentiation is essential in order to establish physiological function of the antigen. One way to illustrate in situ synthesis of the antigen is to demonstrate the presence of messenger RNA (mRNA) which encodes the amino acid sequence of the antigen. To localize a particular mRNA at the lavel of cells, recently in situ hybridization techniques are utilized. As a non-radioactive marker for in situ hybridization. We introduced and utilize thymine-thymine dimers. When DNA is irradiated with ultraviolet light, thymine-thymine (T-T) dimer is formed between adjacent thymines. Since the T-T dimer is a well recognized antigen, after hybridization of T-T dimerized DNA with cellular mRNA, the sites of T-T dimer was recognized by routine immunohistochemistry. When the T-T dimerized probe is used, the resolution is high and intracellular sites of the target may be recognized both at the light and electron microscopic levels, and the sensitivity may be elevated by immunohistochemical and enzyme-histochemical procedural modifications.