Ultrastructural localization of herpes simplex virus RNA by in situ hybridization.

Wolber, Robert; Beals, Theodore F.; Maassab, Hunein F.

doi:10.1177/37.1.2535704

Cited by 37 publications

(18 citation statements)

References 11 publications

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“…Ultrastructural ISH for mRNA was first described by Jacob et al (1971), and has been further developed by several investigators (Guitteny and Bloch 1989;Jirikowski et al 1990;Le Guellec etal. 1990;Morel et al 1989a, b;Pomeroy et al 1991;Singer et al 1989;Trembleau et al 1990;Webster et al 1987;Wolber et al 1989). Ultrastructural ISH has been carried out in different ways, namely, pre-embedding (Guitteny and Bloch 1989;Pomeroy etal.…”

Section: Discussionmentioning

confidence: 99%

“…Ultrastructural ISH has been carried out in different ways, namely, pre-embedding (Guitteny and Bloch 1989;Pomeroy etal. 1991;Trembleau et al 1990;Wolber et al 1989), ultrathin frozen sections (Morel et al 1989a, b), and post-embedding (Jirikowski et al 1990;Le Guellec et al 1990;. On the basis of our assessment of ultrastructural preservation reported previously (Matsuno et al 1993;1994a), we used both pre-and post-embedding EM-ISH in this study.…”

Section: Discussionmentioning

confidence: 99%

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Changes in the ultrastructural distribution of prolactin and growth hormone mRNAs in pituitary cells of female rats after estrogen and bromocriptine treatment, studied using in situ hybridization with biotinylated oligonucleotide probes

Matsuno

Ohsugi

Utsunomiya

et al. 1995

Histochem Cell Biol

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The expression and distribution of prolactin (PRL) mRNA and their alterations induced by estrogen and bromocriptine were investigated using non-radioisotopic in situ hybridization (ISH) at the electron microscopic (EM) level. Our EM-ISH studies using biotinylated oligonucleotide probes showed that estrogen induced whirling changes of the rough endoplasmic reticulum (RER) of female rat PRL cells and increased transcription of PRL genes located on the polysomes of the whirling RER. The presence of mammosomatotroph cells in the rat pituitary gland was also verified in our EM-ISH studies. After bromocriptine administration, PRL cells contained many secretory granules due to the inhibition of secretion. Pre- and post-embedding EM-ISH and northern hybridization studies revealed that bromocriptine induced the distorted, vesiculated, and dilated RER, and also the suppressed PRL mRNA expression. The activity of protein kinase C (PKC), which mediates PRL gene expression, tended to be elevated by estrogen and suppressed by bromocriptine. Therefore, it is considered that the ultrastructural and quantitative changes in PRL mRNA expression evoked by estrogen and bromocriptine may be mediated by the intracellular signal transduction system, including PKC.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Changes in the ultrastructural distribution of prolactin and growth hormone mRNAs in pituitary cells of female rats after estrogen and bromocriptine treatment, studied using in situ hybridization with biotinylated oligonucleotide probes

Matsuno

Ohsugi

Utsunomiya

et al. 1995

Histochem Cell Biol

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“…Labeling is restricted to those specific nucleic acid sequences which are exposed at the surface of the section; those which are within the interior of the section are unlabeled. Nevertheless, permeabilizing of the cells which is necessary to allow penetration of the probe and its subsequent labeling in pre-embedding hybridization (Wolber et al, 1988(Wolber et al, , 1989) is eliminated. Therefore, the post-embedding procedure used in this report, which better preserves morphological detail, is the most useful method for identifying with certainty the structures which are specifically labeled.…”

Section: Discussionmentioning

confidence: 99%

“…It is now markedly improved by labeling of the probes with a biotinylated nucleotide and its subsequent detection with cytochemical techniques (Binder et al, 1986;Escaig-Haye et al, 1989;Hutchison et al, 1982;Manuelidis et al, 1982;McFadden, 1989;Puvion-Dutilleul and Puvion, 1989;Thiry and Thiry-Blaise, 1989;Wolber et al, 1988Wolber et al, , 1989.…”

Section: Introductionmentioning

confidence: 99%

Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization of biotinylated DNA probes on sections of herpes simplex virus type 1 infected cells

Puvion‐Dutilleul

Puvion

1991

J. Elec. Microsc. Tech.

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The influence of fixation and enzymatic digestions on the ability of a denatured double-stranded DNA probe to bind specifically to related sequences of RNA and DNA in sections of Lowicryl embedded cells was investigated. Specificity of the hybridization was assessed using a biotinylated cloned subgenomic herpes simplex virus type 1 DNA fragment to localize viral nucleic acids in sections of infected cells. The probe was detected by anti-biotin antibodies and indirect immunogold labeling. Controls indicated that protease digestion of proteins from the section eliminated non-specific binding of the probe and labeling of endogenous biotin. Both formaldehyde and glutaraldehyde fixation retained viral RNA in protease digested sections. Its labeling was randomly and sparsely distributed over the fibrillo-granular network of the infected nucleus and over the ribosome-rich regions of cytoplasm. Labeling of single-stranded portions of viral DNA in protease-RNase digested sections was infrequent. It was located precisely over nucleoids of a few viral nucleocapsids whatever their location in the cell and their stage of maturation. Labeling of double-stranded viral DNA by denaturation of the DNA in the sections of Lowicryl embedded cells was possible after fixation with formaldehyde but not glutaraldehyde. Among several denaturation protocols, 0.5 N NaOH treatment was best for hybridization of both non-encapsidated and encapsidated viral DNA in protease-RNase digested sections. Free viral genomes were detected exclusively within the virus-replicating region of infected nuclei. Labeling of viral nucleoids was independent of their location in the cell. The high percentage of labeled viral nucleoids suggests that the related viral DNA sequence is not aggregated in the nucleoid but is extended and therefore numerous portions of this defined DNA sequence are accessible at the surface of the section for the binding of the probe.

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“…In addition, antibodies conjugated to different sizes of gold particles allow, in principle, double immunolabeling on the same specimen. However, to gain ac cess for immunogold labels, cells often have to be permeabilized with proteolytic enzymes or detergents, and such treatments can com prom ise cell morphology to an unacceptable extent (Webster et al 1987;Wolber et al 1989;Lloyd et al 1990), even if F(ab)2 fragments ad sorbed to ultra-small colloidal gold (Sibon et al 1994(Sibon et al , 1995Macville et al 1995) or covalently bound to goldatom clusters (Huang et al 1994) are used. Alternatively, post-embedding EM ISH methods allow direct access for nucleic acid probes and gold-conjugated antibodies at the sectioned surface.…”

Section: Introductionmentioning

confidence: 99%

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

Macville

Dorp

Dirks

et al. 1996

Histochem Cell Biol

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The in situ hybridization (ISH) technique, as applied to electron microscopic detection of RNAs, was evaluated for ultra-thin cryosections of cultured rat fibro blasts (rat 9G). Experimental variables to balance penetra tion of detection reagents and preservation of ultrastructural morphology included various strengths of aldehyde fixation and pepsin treatment. We performed ISH for 28S ribosomal RNA (rRNA) followed by ultra-small colloidal gold immunocytochemistry and silver enhancement. An acceptable balance for 28S rRNA ISH detection was ob tained using mild cross-linking fixation followed by treat ment with a relative high concentration of pepsin for a short time. The ISH method presented in this study was compatible with immunocytochemical detection of protein as demonstrated by double-labeling experiments.

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Ultrastructural localization of herpes simplex virus RNA by in situ hybridization.

Abstract: and 17.5 (33% increase), respectively.

Cited by 37 publications

References 11 publications

Changes in the ultrastructural distribution of prolactin and growth hormone mRNAs in pituitary cells of female rats after estrogen and bromocriptine treatment, studied using in situ hybridization with biotinylated oligonucleotide probes

Changes in the ultrastructural distribution of prolactin and growth hormone mRNAs in pituitary cells of female rats after estrogen and bromocriptine treatment, studied using in situ hybridization with biotinylated oligonucleotide probes

Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization of biotinylated DNA probes on sections of herpes simplex virus type 1 infected cells

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

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