scholarly journals Enhanced sensitivity for light and electron microscopic in situ hybridization with multiple simultaneous non-radioactive oligodeoxynucleotide probes.

1995 ◽  
Vol 43 (8) ◽  
pp. 829-841 ◽  
Author(s):  
A Trembleau ◽  
F E Bloom
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent Dyes ◽  
General Procedure ◽  
Simultaneous Detection ◽  
High Sensitivity ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Enhanced Sensitivity ◽  
Low Sensitivity

Although oligonucleotide probes are useful for in situ hybridization, their low sensitivity compared to riboprobes and cDNA remains a problem. We have systematically examined the protocols to provide a general procedure that increases the sensitivity of oligoprobes for light and electron in situ hybridizations by using mixtures of multiple non-overlapping oligonucleotides (multi-oligoprobes). The protocol achieves these improvements with both radioactive and non-radioactive oligoprobes. With 33P-labeled probes in a semiquantitative assay, we found that mixtures of up to six vasopressin-directed multi-oligoprobes, each employed at saturating concentration, led to an additive signal with no significant increase of the background. Using this approach with non-radioactive oligoprobes, we were able to detect in the hypothalamus several low or moderately abundant mRNAs, such as vasopressin heterogeneous nuclear RNA and the galanin, dynorphin, and tyrosine hydroxylase mRNAs. Moreover, we showed that multi-oligoprobes used in a pre-embedding procedure were suitable for studying the ultrastructural compartmentalization of moderately abundant mRNAs. Finally, with the same basic approach we demonstrated that two sets of multi-oligoprobes can be combined for simultaneous detection of two different mRNAs using fluorescent dyes, making this approach suitable for high-resolution confocal analyses. Overall, our data demonstrate that multi-oligoprobes provide a sensitive tool of choice for various applications in which both well-preserved morphology and high sensitivity are needed. In particular, these probes appear ideal for study of the comparative subcellular localization of mRNAs at both the light and the electron microscopic level.

In Situ Hybridization at the Electron Microscopic Level Using a Bromodeoxyuridine Labeled DNA Probe

1993 ◽  
Vol 68 (3) ◽  
pp. 169-174 ◽  
Author(s):  
Chong-Hua Yao ◽  
Sohei Kitazawa ◽  
Takahiro Fujimori ◽  
Sakan Maeda
Keyword(s):  
In Situ Hybridization ◽  
Microscopic Level ◽  
Dna Probe ◽  
Electron Microscopic Level ◽  
Electron Microscopic

Epstein-Barr Virus (EBV)-Encoded RNA: Automated In-Situ Hybridization (ISH) Compared with Manual ISH and Immunohistochemistry for Detection of EBV in Pediatric Lymphoproliferative Disorders

2009 ◽  
Vol 12 (3) ◽  
pp. 195-199 ◽  
Author(s):  
Naim K. Fanaian ◽  
Cynthia Cohen ◽  
Sandra Waldrop ◽  
Jennifer Wang ◽  
Bahig M. Shehata
Keyword(s):  
In Situ Hybridization ◽  
Predictive Value ◽  
Epstein Barr Virus ◽  
High Sensitivity ◽  
High Specificity ◽  
Lymphoproliferative Disorders ◽  
Barr Virus ◽  
Epstein Barr ◽  
Low Sensitivity

Detection of Epstein-Barr virus (EBV) may be achieved by various methods, including EBV-encoded RNA (EBER) in-situ hybridization (ISH) and immunohistochemistry (IHC) for latent membrane protein (LMP-1). We compared novel automated ISH and IHC techniques in pediatric lymphoproliferative disorders with results obtained by manual ISH. Thirty-seven pediatric cases previously studied by manual EBER ISH (including 18 EBER-positive, 15 EBER-negative, and 4 EBER-equivocal cases) were used for the study. Automated EBER ISH and automated LMP-1 IHC were performed using the BondMax autostainer and prediluted EBER probe and EBV cell surface 1 to 4 at 1:50 dilution, respectively. Results of each of the automated techniques for EBV detection were compared with results by manual EBER ISH. Compared with manual EBER ISH as the gold standard, automated ISH had a sensitivity and specificity of 94% and 69%, respectively, accuracy of 83%, positive predictive value (PPV) of 79%, and negative predictive value (NPV) of 90%. Automated IHC had a sensitivity of 44%, specificity of 93%, accuracy of 67%, PPV of 88%, and NPV of 59%. Automated ISH and IHC correlated significantly ( P < 0.045). Automated ISH is useful for diagnosis of EBV-related pediatric neoplasms, being easy to perform and interpret and requiring only the technologist's time to set up and having a high sensitivity and NPV. The automated IHC protocol is of too low sensitivity for routine use, although results show high specificity and PPV.

Recent Advances in Microwave-Assisted Specimen Processing for in-Situ Hybridization

2001 ◽  
Vol 7 (S2) ◽  
pp. 1198-1199
Author(s):  
Mark A. Sanders ◽  
David M. Gartner
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent Dyes ◽  
High Sensitivity ◽  
Tissue Level ◽  
Antigen Retrieval ◽  
Intact Cells ◽  
Thin Sections ◽  
Specimen Processing ◽  
Catalyzed Reporter Deposition

Co-localization of proteins and nucleic acid sequences by in situ hybridization and immunohistochemistry is frequently difficult as the procedure necessary to detect the target structure of one technique may negatively affect the target of the other. Structural impairments may also limit the application of the two techniques. to overcome these problems we developed a method to perform in situ hybridization and immunohistochemistry on Chromosome spreads, intact cells and semi-thin sections of methyl methacrylate-embedded tissue. Microwavestimulated antigen retrieval, signal amplification by catalyzed reporter deposition, and fluorescent dyes were used for all techniques, yielding high sensitivity and excellent morphological preservation compared to conventional processing techniques. Co-localization of in situ hybridization and immunohistochemistry probes with high morphological resolution was achieved using computerized image reconstruction. The latter may allow for the co-localization of multiple antigens and a specific DNA sequence at the same tissue level.

scholarly journals Ultrastructural localization of mRNA encoding for the EGF receptor in human breast cell cancer line BT20 by in situ hybridization.

1991 ◽  
Vol 39 (1) ◽  
pp. 1-6 ◽  
Author(s):  
D Le Guellec ◽  
L Frappart ◽  
P Y Desprez
Keyword(s):  
In Situ Hybridization ◽  
Cell Line ◽  
Egf Receptor ◽  
Cell Cancer ◽  
Electron Microscopic Level ◽  
Synthesis Process ◽  
R Gene ◽  
Electron Microscopic ◽  
Quick Freezing

The EGF receptor (EGF-R), a 170 KD transmembrane glycoprotein, is found at a high level in the BT20 human mammary carcinoma cell line (1 +/- 0.4 x 10(6) sites per cell). In this study, we examined the expression of the EGF-R gene in BT20 cell line by in situ hybridization at the light and electron microscopic level using a human cDNA, corresponding to EGF-R transmembrane and protein kinase domains, labeled with [3H]-, [35S]-, or [32P]-d-ATP. Two treatments were tested to embed cells in Lowicryl resin: the first used fixation and dehydration by progressive lowering of temperature, the second quick freezing and cryosubstitution. The best ultrastructural preservation was obtained with the second procedure without modification of the hybridization signal. EGF-R mRNA was observed principally at the cytoplasmic level, on organelles involved in the protein synthesis process. Labeling was also located on the microvilli which extend into the intercellular space, suggesting that some mRNA would be located in sites where EGF-R is utilized. Some mRNA was observed in the nucleus. This study demonstrates that post-embedding in situ hybridization, after quick freezing and cryosubstitution, is a powerful EM in situ hybridization procedure to study the expression of the EGF-R gene.

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