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This is a presentation of Exiqons miRCURY LNA™ microRNA Detection Probes for In Situ Hybridization.
Locked nucleic acid (LNA™) nucleosides are a class of nucleic acid analogues in which the ribose ring is “locked” by a methylene bridge connecting the 2’-O atom and the 4’-C atom. LNA™ nucleosides contain the six common nucleobases (T, C, G, A, U and mC) that appear in DNA and RNA and are able to form base pairs according to standard Watson-Crick base pairing rules. However, by “locking” the molecule with the methylene bridge the LNA™ is constrained in the ideal conformation for Watson-Crick binding. When incorporated into a DNA oligonucleotide, LNA™ therefore makes the pairing with a complementary nucleotide strand more rapid and increases the stability of the resulting duplex.
LNA™ Oligonucleotides
An LNA™-enhanced oligonucleotide is the ideal choice whenever short or very similar sequences need to be analyzed. The high affinity of an LNA™ oligonucleotide to its complementary sequence results in higher specificity and sensitivity than when using traditional DNA or RNA sequences. In many cases, LNA™-enhanced oligonucleotides can distinguish between sequences differing by a single nucleotide, which can be critical for the success of many experiments.
Posted by admin on Friday, February 20 @ 08:55:25 CET (1814 reads) (Read More... | 3252 bytes more | Score: 0)
Protocols
Cold Spring Harbor Protocols
Cold Spring Harbor Laboratory is renowned for its teaching of biomedical research techniques. For decades, participants in its celebrated, hands-on courses and users of its laboratory manuals have gained access to the most authoritative and reliable methods in molecular and cellular biology. Now that access has moved online.
Cold Spring Harbor Protocols is a definitive, interactive source of new and classic research techniques. The database is fully searchable by keyword and subject, and it has many novel features—such as discussion forums and personal folders—made possible by online publication. Its coverage includes cell and molecular biology, genetics, bioinformatics, protein science, and imaging. Protocols are presented step-by-step and edited in the style that has made Molecular Cloning, Antibodies, Cells and many other CSH manuals essential to the work of scientists worldwide. Protocols will be continuously expanded, updated, and annotated by the originators and users of the techniques.
Posted by admin on Wednesday, January 07 @ 11:18:30 CET (1633 reads) (Read More... | Score: 0)
GEISHA
Gallus Expression In Situ Hybridization Analysis
The online repository for chicken in situ hybridization information. This site presents whole mount in situ hybridization images and corresponding probe and genomic information for genes expressed in chicken embryos.
The goals of the GEISHA project are to obtain whole mount in situ hybridization expression information for all differentially expressed genes in the chicken embryo between HH stages 1-25, to integrate expression data with the chicken genome browsers, and to offer this information through a user-friendly graphical user interface.
Posted by admin on Monday, January 05 @ 11:19:26 CET (1579 reads) (Read More... | 976 bytes more | Score: 0)
EnzMet (SISH)
Review Paper Describes Development and Use of New Enzyme Metallography (EnzMetTM) Reagent for In Situ Hybridization
Nanoprobes, Incorporated has developed a new system for super-sensitive, high-resolution detection for use in in situ hybridization (ISH) and immunohistochemistry (IHC). The new process uses an enzyme-linked probe to deposit metal from solution at a target site; this provides a dense, punctate, high resolution black stain which is readily distinguished from other commonly used stains. This new detection method, termed "Enzyme Metallography" (EnzMetTM) has several important advantages for pathology and tissue study. It is visualized in the conventional brightfield light microscope, and hence does not require the use of fluorescent optics or dark adaptation on the part of the user. It provides a permanent record, with no fading or photobleaching; and it can visualize single copies of even non-amplified genes.
The development of this method and its application to HER2 gene amplification in breast cancer are described in a review in the August issue of Human Pathology. Nanoprobes recently signed a deal with Ventana Medical Systems, Incorporated, for commercial development and use of this reagent in their automated slide staining instruments. As a result, the first commercial product has now been introduced in Europe; it is called SISH (Silver In Situ Hybridization). Introduction of SISH in the United States is pending FDA approval. In addition, Nanoprobes will shortly introduce a commercial EnzMetTM formulation optimized for research applications and non-automated staining.
Reference:
Powell, R. D.; Pettay, J. D.; Powell, W. C.; Roche, P. C.; Grogan, T. M.; Hainfeld, J. F., and Tubbs, R. R.: Metallographic in situ hybridization. Hum. Pathol., 38, 1145-1159 (2007). Article (pdf)
Posted by admin on Monday, December 03 @ 15:11:03 CET (9419 reads) (Read More... | Score: 3.66)
Multiple Labeling
A Novel Method for Multiple Labeling Combining In Situ Hybridization With Immunofluorescence
In situ hybridization (ISH) is a particularly useful method to investigate de novo mRNA expression in tissue sections. High specificity and sensitivity of this technique combined with the great preservation of tissue and cellular morphology conferred by fixatives such as 4% paraformaldehyde, pH 9.5, make ISH a tool of choice for detecting genes of interest in individual cells in the central nervous system (CNS). Here we describe a novel method that combines radioactive ISH with immunofluorescence on the same tissue section to identify cell populations expressing selected mRNA transcripts. This novel method has several major advantages over previously described double-labeling light microscopic methods combining enzymatic immunohistochemistry and ISH including complete protection against loss of hybridization signal that normally occurs during the immunoenzymatic reaction, improved immunolabeling sensitivity due to the proteinase K digestion step during ISH, detection of several proteins specific for different cell populations on the same tissue section, and counterstaining of tissue sections without affecting visualization of immunolabeling. This new method will be particularly useful for investigators looking to identify cell populations producing mRNAs expressed in low abundance such as cytokines, chemokines, and growth factors in the intact and/or injured mammalian CNS.(J Histochem Cytochem 54:1303–1313, 2006)
Posted by admin on Sunday, November 05 @ 13:17:36 CET (2635 reads) (Read More... | Score: 5)
In Situ Hybridization
Roche Diagnostics GmbH and GeneDetect finalize worldwide licensing agreement.
Dr Paul Hughes, CEO of GeneDetect. "Today I am happy to announce that GeneDetect has negotiated a broad license to the valuable Roche Diagnostics GmbH, Digoxigenin/Digoxin patent portfolio for the detection of nucleic acids. This license will allow GeneDetect to release a range of new products incorporating Digoxigenin/Digoxin. We expect these products to become available early in 2006".
Posted by admin on Sunday, March 26 @ 09:10:55 CEST (3545 reads) (Read More... | Score: 3.66)
Polysomy of chromosome 17 in breast cancer
Polysomy of chromosome 17 in breast cancer tumors showing an overexpression of ERBB2: a study of 175 cases using fluorescence in situ hybridization and immunohistochemistry
Abstract
Introduction
One of the most common genetic aberrations associated with breast cancer is the amplification and overexpression of the ERBB2 proto-oncogene located at chromosome 17, bands q12-21. The amplification/overexpression occurs in 25 to 30% of all breast cancers. In breast cancer, aneusomy of chromosome 17, either monosomy or polysomy, is frequently observed by conventional cytogenetics and fluorescence in situ hybridization (FISH). The aim of this study was to discover whether or not numerical aberrations on chromosome 17 have a correlation to the amplification or overexpression of the ERBB2 gene and to analyze their clinical implications in subgroups showing 2+ or 3+ positive scores by immunohistochemistry (IHC).
Methods
We used FISH on a series of 175 formalin-fixed paraffin-embedded breast carcinomas to detect ERBB2 amplification, using a dual-probe system for the simultaneous enumeration of the ERBB2 gene and the centromeric region of chromosome 17, as well as using IHC to detect overexpression. We analyzed clinical and pathological variables in a subgroup of patients with 2+ and 3+ IHC scores (147 patients), to describe any differences in clinicopathological characteristics between polysomic and non-polysomic cases with the use of the χ2 test.
Results
We found 13% of cases presenting polysomy, and three cases presented monosomy 17 (2%). According to the status of the ERBB2 gene, instances of polysomy 17 were more frequently observed in non-amplified cases than in FISH-amplified cases, suggesting that the mechanism for ERBB2 amplification is independent of polysomy 17. Polysomy 17 was detected in patients with 2+ and 3+ IHC scores. We found that nodal involvement was more frequent in polysomic than in non-polysomic cases (P = 0.046).
Conclusions
The determination of the copy number of chromosome 17 should be incorporated into the assesment of ERBB2 status. It might also be helpful to differentiate a subgroup of breast cancer patients with polysomy of chromosome 17 and overexpression of ERBB2 protein that probably have genetic and clinical differences.
Keywords: breast cancer, ERBB2 gene, fluorescence in situ hybridization, immunohistochemistry, polysomy 17
Posted by admin on Tuesday, March 14 @ 08:29:31 CET (2356 reads) (Read More... | 30794 bytes more | Score: 5)
Chromogenic in situ hybridization (CISH)
Chromogenic in situ hybridization (CISH): a novel alternative in screening archival breast cancer tissue samples for HER-2/neu status
Abstract
Background
Chromogenic in situ hybridization (CISH) is emerging as a practical, cost-effective, and valid alternative to fluorescent in situ hybridization in testing for gene alteration, especially in centers primarily working with immunohistochemistry (IHC).
Methods
We assessed Her-2/neu alteration using CISH on formalin-fixed paraffin-embedded primary invasive ductal carcinoma tumors in which IHC (CB11 antibody) had previously been performed, and we compared the results with IHC. The 160 selected cases were equally stratified randomly into the four IHC categories (scores of 0, 1+, 2+, and 3+). We also compared age at diagnosis and tumor histologic grade with IHC and CISH Her-2/neu.
Results
We were able to perform and evaluate CISH successfully on all cases. The agreement between 3+ IHC and CISH-amplified cases as well as between all IHC and CISH Her-2/neu negative cases was 100%, and the concordance on all positive cases was 72.50%, with an overall agreement of 86.25%. All the discordant cases had 2+ IHC scores. Although we noted Her-2/neu positivity more in premenopausal women, the age at diagnosis was not significantly associated with IHC or CISH results. Similarly, although the small group of well-differentiated tumors was apparently Her-2/neu negative in both tests, no significant association was noted between any tumor histologic grade and either IHC or CISH results.
Conclusions
CISH is easily integrated into routine testing in our laboratory. It is a necessary adjunct in determining the subset of non-amplified IHC-positive invasive tumors that will not benefit from trastuzumab therapy. Those cases with 2+ IHC results will be triaged and subjected to CISH. Her-2/neu testing should be done on all breast cancer cases regardless of age at presentation and tumor histologic grade.
Posted by admin on Tuesday, March 07 @ 08:52:33 CET (1487 reads) (Read More... | 35226 bytes more | Score: 5)
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S100 A/B in Marmoset FFPW section of colon: ab14849
Cold Spring Harbor Laboratory is renowned for its teaching of biomedical research techniques. For decades, participants in its celebrated, hands-on courses and users of its laboratory manuals have gained access to the most authoritative and reliable methods in molecular and cellular biology. Now that access has moved online.
The online repository for chicken in situ hybridization information. This site presents whole mount in situ hybridization images and corresponding probe and genomic information for genes expressed in chicken embryos.
![[Enzyme Metallography: schematic and comparison with DAB (74k)]](http://www.nanoprobes.com/Images/News07_ENZ_Fig1.jpg)