Simultaneous ultrastructural identification of growth hormone and its messenger ribonucleic acid using combined immunohistochemistry and non-radioisotopicin situ hybridization: A technical note

Abstract

The present electron microscopical study is concerned with the simultaneous visualization of messenger ribonucleic acid (mRNA) and its encoded protein in the same specimen. Pre-embedding electron microscopical in situ hybridization (EM-ISH) on rat pituitary gland tissue localized growth hormone mRNA in the polysomes of the rough endoplasmic reticulum, and subsequent postembedding immunolabelling using protein A-colloidal gold particles identified growth hormone mainly in the secretory granules. We believe that our report provides the first simultaneous ultrastructural identification of mRNA and its encoded protein using combined pre-embedding EM-ISH and immunohistochemistry. In this method, the signals for mRNA were localized specifically as highly electron dense products on the polysomes of the endoplasmic reticulum, and those for its encoded protein were recognized as gold particles both in the cisternae of the reticulum and in the secretory granules. Our ultrastructural double labelling method for mRNA and protein may provide a tool to find important clues for elucidating the intracellular correlation of mRNA translation and secretion of translated protein, because of its high resolution, good morphological preservation, and the specific localization of the reaction products.

Two- or Three-Dimensional Imagings of Simultaneous Visualization of Rat Pituitary Hormone and Its mRNA: Comparison between Electron Microscopy and Confocal Laser Scanning Microscopy with Semiconductor Nanocrystals (Quantum dots)

“…As shown in our previous reports [14][15][16][17][18][19], EM-ISH with an antisense probe for rat GH mRNA reveals its localization on the rough endoplasmic reticulum (RER) (Fig. 1).…”

“…The detailed method of EM-ISH&IHC was described in our previous reports [14][15][16][17][18][19]. Briefly, hybridization was carried out using biotinylated oligonucleotide probes.…”

Molecular Morphology of Pituitary Cells, from Conventional Immunohistochemistry to Fluorescein Imaging

“…We developed a non-radioisotopic EM-ISH method using biotinylated synthesized oligonucleotide probes, and applied this method to the ultrastructural visualization of growth hormone (GH) and prolactin (PRL) mRNAs and pathophysiological studies in rat pituitary cells [ 1 , 2 , 3 ]. In addition, we developed a combined EM-ISH and immunohistochemistry (IHC) (EM-ISH&IHC) technique for the purpose of simultaneously identifying pituitary hormone and its message in the same cell [ 4 , 5 , 6 ]. This method has been utilized to investigate the intracellular localization of pituitary hormone and its mRNA at the same time [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ].…”

Co-labeling Using In Situ PCR

“…In addition, most recommend doing the ISH analysis first, followed by IHC. Finally, Matsuno et al (1996) have reported on a protocol allowing co-localization of an mRNA and a protein after electron microscopy-based ISH and IHC.…”