In situ hybridization applications

In the tissue sections, a hybridization is performed with a highly specific probe in order to detect the target (either amplified by indirect in situ RT-PCR or unamplified). For the preparation of nonradioactive digoxigenin-labeled probes, one should refer to the guidelines of the company manuals Genius M System User s guide for membrane hybridization and Nonradioactive in situ hybridization application manual (2nd ed., Boehringer Mannheim). 

Nonradioactive in Situ Hybridization Application Manual, Boehringer Mannheim GmbH Biochemica, 1992. A copy of this 75 page booklet may be obtained by writing to PO box 310120, D-6800 Mannheim 31, Germany. 

PNA oligomers have also found widespread application as probes for in situ hybridization (FISH) both in human diagnostics [119-122] and in environmental detection of microbes [123, 124]. 

Regnault, B. Martin-Delautre, S. Lejay-Collin, M. Lefevre, M. Grimont, P. A. D. Oligonucleotide probe for the visualization of Escherichia coli Escherichia fergu-sonii cells by in situ hybridization Specificity and potential applications. Res. Microbiol. 2000,151,521-533. 

EXTENDED APPLICATION OF ANTIGEN RETRIEVAL TECHNIQUE IN IMMUNOHISTOCHEMISTRY AND IN SITU HYBRIDIZATION... 

Recently, the use of AR has extended into several other areas, yielding interesting information for cytology, fresh cell/tissue sections, and fluorescence IHC (fluorescence in situ hybridization [FISH]), in addition to adaptations of the method for extraction of nucleic acids and proteins from FFPE tissues for use with modern methods of molecular analysis. In this chapter, the emphasis is on expanded applications in diagnostic cytology, fresh frozen cell/... 

Sugimura H. Detection of chromosome changes in pathology archives an application of microwave-assisted fluorescence in situ hybridization to human carcinogenesis studies. Carcinogenesis 2008 29 681-687. 

Hicks DG, Longoria G, Pettay J, et al. In situ hybridization in the pathology laboratory general principles, automation, and emerging research applications for tissue-based studies of gene expression. J. Mol. Histol. 2004 35 595-601. 

Jackson, P., Dockey, D.A., Lewis, F.A., and Wells, M. (1990) Application of 1-nm gold probes on paraffin wax sections for in situ hybridization histochemistry./. Clin. Pathol. 43, 810-812. 

In situ hybridization (ISH) permits examination of a wide range of materials, ranging from cells and tissues to metaphase spreads of chromosomes affixed to a slide. Hybridization with specific probes can thus be performed directly on the slide, with the hybridized signal viewed under a microscope. Briefly, the steps involved in ISH include pretreatment of the slide, application of the sample, fixing of the sample, hybridization with probe, and visualization of the probe signal. 

In situ hybridization (ISH) consists of the application of hybridization techniques to intact cells which demonstrate genetic information within a morphologic context. This technology takes advantage of the hybridization properties of nucleic acids and offers a distinct technique to directly analyze sequence information in intact tissues. In essence, it combines cytogenetic techniques with molecular biology to probe gene alterations at molecular levels. Development of... 

Noncovalent interactions such as van der Waals, hydrogen bonding, n-n stacking and electrostatic interactions have been widely used to hybridize pristine nanocarbons via ex situ approaches. The major advantage of this route is that the nanocarbons do not require modification prior to hybridization and their structure remains undisturbed, an important factor in many electronic applications. The strength of hybridization is weaker compared to covalent interactions but the synthetic process is generally simpler. Noncovalent attachment of small molecules to nanocarbons is often used to change the surface chemistry for subsequent ex situ or in situ hybridization. 

MuUink H, Vos W, Jiwa M, Horstman A, der Valk van P, Walboomers JM, Meijer CJ. 1992. Application and comparison of silver intensification methods for the diaminoben-zidine and diaminobenzidine-nickel endproduct of the peroxidation reaction in immunohistochemistry and in situ hybridization. J Histochem Cytochem 40 495-504. 

Table 1 lists representative examples of the application of in situ hybridization, including FISH, to studies that use chromosome preparations and interphase nuclei (4,5). FISH is superior to in situ hybridization with an enzymatic probe because it provides finer resolution and higher signal intensity. This is especially important when the localization of a gene within a specific chromosome band is to be established. 

Applications of In Situ Hybridization to Chromosomes and Interphase Nuclei... 

Nuovo, G. J. (1997) PCR in situ hybridization protocols and applications, 3rd ed. Lippincott-Raven, Philadelphia, PA. 

Another important application of cDNAs is to identify specific proteins in a tissue homogenate or tissue section. Since cDNAs undergo complementary base pairing, adding a radioactively labelled cDNA to a homogenate or tissue slice will bind it to the complementary sequence by a process of hybridization. Thus the amount of radioactive cDNA that hybridizes to the tissue or tissue extract is a measure of the amount of mRNA that is complementary to it. When this procedure is undertaken on slices of brain, it is known as in situ hybridization. In this way it is possible to determine the distribution of specific receptors in a tissue by accurately determining the distribution of mRNA that encodes for the receptor protein. This is a particularly valuable technique for the administration of psychotropic drugs. 

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