Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding.

Abstract

In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowiayl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-2ldUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the en- In ttoductionImmunocytological studies have shown that growth hormone (GH) is synthesized in cells of the pituitary gland called somatotrophs and is stored in secretory granules. Although the presence of GHlike immunoreactivity (GH-LI) in somatotrophs has been observed with all the techniques described below, the localization of immunoreactivity was not exactly the same in each case. Using preembedding methods (Dachew and Dubois, 1976) or ultra-thin frozen sections (Hemming et al., 1989), immunoreactivity could be detected in the secretory granules where GH is stored, as well as in the endoplasmic reticulum where GH is synthesized. After embedding, using hydrophilic (Roson and Beiras, 1991) or epoxy (Li et al., 1977;Moriarty, 1973;Nakane, 1970) zymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmollml was suficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmollml of probe) but the lowest ultrastructural preservation. On ultrathin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation. (]Histo&em Cytochem 40:979-986, 1 9 2 )

Application of Ultrastructural In Situ Hybridization Combined with Immunohistochemistry to Pathophysiological Studies of Pituitary Cell: Technical Review.

“…In contrast with the previous report of postembedding study using Lowicryl embedment [9], in which hybridization signals were not located precisely on the polysomes of the RER but localized more frequently on the ER cisternal space, visualization with higher resolution and more correct localization of hybridization signals for rat GH mRNA on the polysomes of the RER can be obtained using LR White resinembedded specimens.…”

“…Ultrastructural ISH for mRNA, which was first described by Jacob et al [5], has further been developed by several investigators [4, 6-9, 11-13, 18-25] and has been carried out in the three different approaches: preembedding method [4, 12-14, 20, 23, 25], ultrathin frozen sections [9,18,19], postembedding method [6-9, 12, 14]. In recent years, we have developed a non-radioisotopic EM-ISH 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 [12][13][14].…”

Evaluation of pepsin treatment for electron microscopic RNA in situ hybridization on ultra-thin cryosections of cultured cells

“…In m ethodological studies for post embedding EM ISH, several em bedding media such as Lowicryl K4 M, LR W hite, LR Gold, and also cryosec tions were evaluated with respect to detection efficiency and preservation of ultrastructural morphology. The highest detection efficiency was found on ultra-thin cryosections using 5-nm gold particles, whereas the u l trastructure was best m aintained in Lowicryl K4M-embedded tissue (Wenderoth and Eisenberg 1991;Dirks et al 1992;Le Guellec et al 1992). Com pared with pre embedding EM ISH, in our opinion the detection effi ciency on cryosections was low.…”

“…In comparative post-embedding EM ISH studies, in which various embedding media and non-embedded frozen ma terials were tested, the most efficient ISH detection was found for ultra-thin cryosectioned tissue using 5-nm col loidal gold (Wenderoth and Eisenberg 1991;Dirks et al 1992;Le Guellec et al 1992). Consequently, the use of ultra-small colloidal gold particles with silver enhance ment on ultra-thin cryosections should yield an even higher detection efficiency.…”

Resin Tissue Microarrays: a Universal Format for Immunohistochemistry

“…Although resin TMAs have not been processed using this method, there is evidence that fixation by formalin or glutaraldehyde and embedding in acrylate resin allows the demonstration of DNA and mRNA transcripts (Le Guellec et al 1992;Morey et al 1995).…”