A combined in situ hybridization (ISH) and immunohistochemistry (IHC) under electron microscopy (EM-ISH&IHC) has sufficient ultrastructural resolution, and provides two-dimensional images of subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (Quantum dots, Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of subcellular localization of pituitary hormone and its mRNA. Both EM-ISH&IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationship between protein and mRNA simultaneously in two or three dimensions. Another important issue is the intracellular transport and secretion of pituitary hormone. We have developed an experimental pituitary cell line, GH3 cell, which has growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes GH linked to EYFP upon stimulated by Ca2+ influx or Ca2+ release from storage. This GH3 cell is useful for the real-time visualization of the intracellular transport and secretion of GH. These three methods enable us to visualize consecutively the process of transcription, translation, transport, and secretion of pituitary hormone.
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