scholarly journals Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

1992 ◽  
Vol 40 (11) ◽  
pp. 1647-1657 ◽  
Author(s):  
R W Dirks ◽  
A G Van Dorp ◽  
J Van Minnen ◽  
J A Fransen ◽  
M Van der Ploeg ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Lymnaea Stagnalis ◽  
Pond Snail ◽  
28S Rrna ◽  
Electron Microscopic ◽  
Rna Sequences ◽  
Double Labeling ◽  
Thin Sections ◽  
Cell Hormone

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.

scholarly journals Saponin pre-treatment in pre-embedding electron microscopic in situ hybridization for detection of specific RNA sequences in cultured cells: a methodological study.

1995 ◽  
Vol 43 (10) ◽  
pp. 1005-1018 ◽  
Author(s):  
M V Macville ◽  
K C Wiesmeijer ◽  
R W Dirks ◽  
J A Fransen ◽  
A K Raap
Keyword(s):  
In Situ Hybridization ◽  
Cultured Cells ◽  
Elongation Factor ◽  
Housekeeping Gene ◽  
28S Rrna ◽  
Electron Microscopic ◽  
Rna Sequences ◽  
Gene Transcripts ◽  
Pre Treatment

We describe a method for detection of specific RNA targets in cultured cells at the electron microscopic (EM) level using pre-embedding in situ hybridization (ISH). The specimens were monitored by reflection-contrast microscopy (RCM) before processing for EM. A good balance between preservation of ultrastructure and intensity of hybridization signals was obtained by using mild aldehyde fixation followed by saponin permeabilization. Digoxigenin-labeled probes were used for detection of human elongation factor (HEF) mRNA in HeLa cells, immediate early (IE) mRNA in rat 9G cells, and 28S rRNA in both cell lines. The hybrids were detected immunocytochemically by the peroxidase/diaminobenzidine (DAB) method or by ultra-small gold with silver enhancement. Comparison of these methods favored the peroxidase/DAB system. The accessibility of RNA in the different cell compartments was dependent on the extent of cross-linking during primary fixation even after permeabilization with saponin. By using the most optimal ISH protocol and the peroxidase/DAB system, we detected 28S rRNA over all ribosomes in the cytoplasm but not in the nucleoli, and IE mRNA in a large spot with many smaller spots around it in the nucleoplasm as well as in speckles over the cytoplasm. The sensitivity of the method is such that HEF housekeeping gene transcripts were detected in the cytoplasm.

scholarly journals Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection.

1989 ◽  
Vol 37 (1) ◽  
pp. 7-14 ◽  
Author(s):  
R W Dirks ◽  
A K Raap ◽  
J Van Minnen ◽  
E Vreugdenhil ◽  
A B Smit ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Lymnaea Stagnalis ◽  
Egg Laying ◽  
Pond Snail ◽  
Specific Staining ◽  
Mrna Detection ◽  
Model Study ◽  
Pre Treatment ◽  
Hybridization Protocol

To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.

Demonstration and Distribution of HCV RNA Sequences by in situ Hybridization and HCV-Related Proteins by Immunohistochemistry in the Liver Tissue of Patients with Chronic HCV Infection

Pathobiology ◽  
1995 ◽  
Vol 63 (5) ◽  
pp. 239-248 ◽  
Author(s):  
Domenico Sansonno ◽  
Vito Cornacchiulo ◽  
Anna Rina Iacobelli ◽  
Pietro Gatti ◽  
Maria Di Stasi ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Liver Tissue ◽  
Hcv Infection ◽  
Hcv Rna ◽  
Rna Sequences ◽  
Chronic Hcv Infection ◽  
Chronic Hcv ◽  
Related Proteins

scholarly journals Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.

1991 ◽  
Vol 39 (11) ◽  
pp. 1495-1506 ◽  
Author(s):  
P M Motte ◽  
R Loppes ◽  
M Menager ◽  
R Deltour
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Spatial Organization ◽  
Higher Plants ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Thin Sections ◽  
Cytoplasmic Dna ◽  
Immunocytochemical Techniques

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.

Genomic in situ hybridization to sectioned nuclei shows chromosome domains in grass hybrids

1990 ◽  
Vol 95 (3) ◽  
pp. 335-341
Author(s):  
A.R. Leitch ◽  
W. Mosgoller ◽  
T. Schwarzacher ◽  
M.D. Bennett ◽  
J.S. Heslop-Harrison
Keyword(s):  
In Situ Hybridization ◽  
Genomic Dna ◽  
Microscope Level ◽  
Hordeum Chilense ◽  
Root Tip ◽  
Interphase Nuclei ◽  
Light Microscope Level ◽  
Thin Sections ◽  
Electron Microscopes

In situ hybridization using biotinylated total genomic DNA and avidin detection systems was adapted for examination of thin-sectioned plant material in the light and electron microscopes. Root tip material was preserved prior to sectioning, so that the in vivo disposition of the chromatin was maintained. Use of total genomic DNA from Secale africanum as a probe enabled the chromatin from the two parental genomes in the grass hybrid Hordeum chilense × S. africanum to be distinguished. The biotinylated probe preferentially labelled the chromosomes of S. africanum origin. DNA-DNA hybrids were visualized at the light-microscope level by Texas Red fluorescence and at the electron-microscope level by the enzymic precipitation of DAB (diaminobenzidine) or by colloidal gold particles. The use of thin sections allowed the location of probe hybridization to be established unequivocally in both metaphase and interphase nuclei. Analysis of interphase nuclei showed that chromatin originating from the two parental genomes did not intermix but occupied distinct domains.

Sign in / Sign up

Export Citation Format

Share Document