Electron Microscopic in Situ Hybridization and its Combination With Immunohistochemistry

Abstract

In situ hybridization at the electron microscopic level is a useful method for examining the intracellular distribution and role of mRNA in protein synthesis. Ultrastructural in situ hybridization for mRNA has been carried out by three different approaches: preembedding method (Guitteny et al. 1989; W olber et al. 1989; Trembleau et al. 1990; Pomeroy et al. 1991; Matsuno et al. 1994a,b, 1995), ultrathin frozen sections (Morel et al. 1989a,b; Le Guellec et al. 1992) and postembedding method (Jirikowski et al. 1990; Le Guellec et al. 1990, 1991, 1992, Matsuno et al. 1994a, 1995). In recent years, we have developed non-radioisotopic electron microscopic in situ hybridization method using biotinylated synthesized oligonucleotide probes for the ultrastructural visualization of growth hormone (GH) and prolactin (PRL) mRNAs in rat pituitary cells, and have applied this method to pathophysiological studies of pituitary cells (Matsuno et al. 1994a,b, 1995). In addition, we have developed combined method of electron microscopic in situ hybridization and immunohistochemistry for simultaneous detection of pituitary hormone and its message in the same cell (Matsumo et al. 1996), which can serve the visualization of intracellular spatial relationship between pituitary hormone and its message at the ultrastructural level.