Application trials of in situ hybridization at the electron microscopic level

Abstract

In situ hybridization (ISH) is a morphology-oriented technique for demonstrating the presence of specific nucleic acid sequences at chromosomal, cytological and histological levels. It is, however, sometimes difficult to recognize specific cell identity, early phase mRNA expression and alternative splicing because of the limited resolution of the light microscope. To overcome this limitation, we developed an improved technique for ISH at the electron microscopic level, in which pre-embedding hybridization with a non-radioactively labeled probe was used, followed by post-embedding immunoglobulin gold colloid staining. By applying this technique, early phase bone morphogenetic protein-3 mRNA in the nuclei and cytoplasm was successfully demonstrated in a differentiating chondrocytic cell lineage. Moreover, with oligo-DNA probes specific for alternative spliced forms of parathyroid hormone-related protein mRNA, we demonstrated such forms in a hyperplastic parathyroid gland attributed to renal failure.