scholarly journals Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization.

1989 ◽  
Vol 108 (6) ◽  
pp. 2343-2353 ◽  
Author(s):  
R H Singer ◽  
G L Langevin ◽  
J B Lawrence
Keyword(s):  
In Situ Hybridization ◽  
High Resolution ◽  
Colloidal Gold ◽  
Messenger Rna ◽  
Signal To Noise Ratio ◽  
Simultaneous Detection ◽  
Gold Particles ◽  
Rna Molecules ◽  
Nucleic Acid Molecule

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.

Ultrastructural analysis of the spatial distribution of MRNA

1992 ◽  
Vol 50 (1) ◽  
pp. 552-553
Author(s):  
Gary Bassell ◽  
Robert H. Singer
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Nucleic Acids ◽  
Colloidal Gold ◽  
Dna Probe ◽  
Nucleotide Analogs ◽  
Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

scholarly journals Electron microscopic location of mRNA in the rat kidney: improved post-embedding in situ hybridization.

1995 ◽  
Vol 43 (8) ◽  
pp. 801-809 ◽  
Author(s):  
J Yi ◽  
O Michel ◽  
C Sassy-Prigent ◽  
J Chevalier
Keyword(s):  
In Situ Hybridization ◽  
Signal To Noise Ratio ◽  
Rat Kidney ◽  
Enzymatic Method ◽  
Electron Microscopic ◽  
Cytoplasmic Matrix ◽  
Gold Particles ◽  
Hybridization Efficiency ◽  
Endogenous Biotin

We determined the optimal technical conditions for post-embedding non-radioactive in situ hybridization applied to ultrastructural location of collagen I mRNA in rat kidney. The signal-to-noise ratio was improved by enhancing hybridization efficiency and distinguishing nonspecific labeling. Probes were labeled with digoxigenin or biotin and detected after hybridization by immunogold or peroxidase techniques. Under these conditions, the signal was located in fibroblasts. With digoxigenin, clusters of gold particles were observed on the endoplasmic reticulum (ER) or scattered throughout the cytoplasmic matrix and nuclei. With the enzymatic method, diaminobenzidine deposits were found on the ER but endogeneous peroxidase partly interfered with the results. Gold particles were less numerous in fibroblast cytoplasm with biotin than with digoxigenin. Moreover, gold particles condensed on fibroblast and tubular cell mitochondria when biotin was used, a phenomenon shown to be due to endogenous biotin by means of a histochemical method. The digoxigenin-immunogold system appeared to be the best method. The biotin system was subject to limitations such as interference from endogenous biotin and poor sensitivity, and mRNA localization was more precise and reliable by the immunogold method than by the enzymatic method.

scholarly journals Demonstration of Balbiani ring RNA sequences in polysomes.

1977 ◽  
Vol 73 (1) ◽  
pp. 260-264 ◽  
Author(s):  
L Wieslander ◽  
B Daneholt
Keyword(s):  
In Situ Hybridization ◽  
Messenger Rna ◽  
Sucrose Gradient ◽  
Balbiani Ring ◽  
Rna Sequences ◽  
Rna Molecules ◽  
Labeled Rna ◽  
Active Genes

A polysome extract from salivary glands of C. tentans was sedimented in a 15-60% sucrose gradient. Fractions from the heavy polysome region (1,000-2,000S) and fractions from the light polysome region (200-1,000S) were pooled separately, and the long-term labeled RNA was released by Sarkosyl/pronase and analysed by in situ hybridization. The results showed that BR 1 and BR 2 sequences were present in the heavy and the light polysome regions of the sucrose gradient. From control experiments with EDTA-treated extracts, it was concluded that most of the recorded BR 1 and BR 2 sequences were in fact located in polysomes. The finding that BR products enter polysomes suggests that they act as messenger RNA molecules. This study therefore strongly supports the concept that chromosome puffs represent active genes.

An automatic analysis method for in situ hybridization using high-resolution image analysis

1989 ◽  
Vol 281 (5) ◽  
pp. 336-341 ◽  
Author(s):  
W. Stolz ◽  
K. Scharffetter ◽  
W. Abmayr ◽  
W. K�ditz ◽  
T. Krieg
Keyword(s):  
Image Analysis ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Automatic Analysis ◽  
Analysis Method ◽  
Resolution Image ◽  
High Resolution Image

High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones

Science ◽  
1990 ◽  
Vol 247 (4938) ◽  
pp. 64-69 ◽  
Author(s):  
P Lichter ◽  
C. Tang ◽  
K Call ◽  
G Hermanson ◽  
G. Evans ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
High Resolution ◽  
Human Chromosome ◽  
Chromosome 11 ◽  
High Resolution Mapping ◽  
Resolution Mapping
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