Application trials of in situ hybridization at the electron microscopic level.
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.
Kitazawa
Sohei
S
Division of Molecular Pathology, Kobe University Graduate School of Medicine, Kusunoki-cho, Chuo-ku, Kobe, Japan. kitazawa@med.kobe-u.ac.jp
Kondo
Takeshi
T
Kitazawa
Riko
R
D017398:Alternative Splicing; D000818:Animals; D055398:Bone Morphogenetic Protein 3; D019485:Bone Morphogenetic Proteins / Q000235:genetics*; D002454:Cell Differentiation; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000648:ultrastructure; D005333:Fetus; D006801:Humans; D006965:Hyperplasia / Q000235:genetics; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D046529:Microscopy, Electron, Transmission; D010280:Parathyroid Glands / Q000378:metabolism / Q000648:ultrastructure; D044162:Parathyroid Hormone-Related Protein / Q000235:genetics*; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*
C525757:BMP3 protein, human; D055398:Bone Morphogenetic Protein 3; D019485:Bone Morphogenetic Proteins; D044162:Parathyroid Hormone-Related Protein; D015347:RNA Probes; D012333:RNA, Messenger