Localization in situ of specific RNA by electron microscopy

To better understand the metabolism of RNA in nuclei, the analysis of preci se nuclear distribution of specific RNA would be essential. For this purpos e, nonradioactive electron microscopic (EM) in situ hybridization may be th e most appropriate technique while the details required for the technique h ave not been fully established. In the present study, we attempted to local ize 28S and 18S rRNAs in the nuclei of mouse Sertoli cells by EM in situ hy bridization as a model system. After various preliminary experiments we cho se the pre-embedding method; fresh-frozen sections of mouse testis were fix ed with a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde, digested with 10 mug/ml of proteinase K and hybridized with thymine-thymine (T-T) di merized oligodeoxy-nucleotides (oligo-DNA) complementary to a part of 28S a nd 18S rRNAs. Then, the T-T dimers were detected enzyme-immunohistochemical ly with horseradish peroxidase (HRP) labeled antiT-T dimer. After osmificat ion of HRP products, the sections were embedded in Epon resin, cut into 100 nm ultra-thin sections and observed under a transmission electron microsco pe. As a result, we successfully localized both 28S and 18S rRNAs in the de nse fibrillar and granular components of the nucleolus, showing the usefuln ess of nonradioactive EM in situ hybridization in the nuclear localization of specific RNA.