Situ Hybridization

In situ hybridization was derived from the techniques of molecular hybridization of nucleic acids that are isolated from a particular cell population or tissue and bound to solid supports. Hybridization of such averaged membrane-bound nucleic acids identifies different classes of DNA (Southern blot) and RNA (Northern blot). However, ISH fills the gap between the detection of a specific sequence and its precise location within the tissue or the cell (Chevalier et al., 1997). 

The role of high temperatures in the breakdown of protein crosslinks introduced by aldehydes has been discussed earlier in this book. The aforementioned combined technique is especially effective in increasing the in situ hybridization signal in archival tissues, the fixation history of which may or may not be known. This protocol also detects low levels of nucleic acids in the tissue, which may not be detectable with heating or enzyme digestion alone. 

In some cases, the use of medium wattages (e.g., 450 W) is preferred over high-power microwave outputs (e.g., 700 W). This difference has been demonstrated in the ISH for detecting measles virus and chicken anemia virus in formalin-fixed, paraffin-embedded brain tissue (McMahon and McQuaid, 1996). In this study higher power outputs resulted in decreased sensitivity. Although the exact reason for this phenomenon is not known, it is hypothesized that optimal oscillation of dipolar molecules produces optimal thermal effects in tissue sections at medium wattages. 

Urea (0.01 M), sodium carbonate (0.01 M), magnesium chloride (0.01 M), or distilled water can be used as microwave fluids to obtain similar results in terms of both sensitivity and intensity of the hybridization signal. Alternatively, 10 mM citrate buffer (pH 6.0) can be used as the microwave fluid. The major role of these fluids is to mediate high temperature effects, which is confirmed by the achievement of a good hybridization signal using distilled water. Note that pretreatment conditions must be optimized for every tissue type and for every cell type in a given section. 

In situ hybridization (ISH) is another important molecular technique allowing the detection of specific nucleic acid sequences within cells or tissue structures using 

Radioactive technique based on nucleic acid probes labeled by radioactive isotopes such as P32, H3, I131 and S35. 

Non-radioactive technique based on the use of non-radioactive-labeled nucleic acid probes. 

Depending on the visualization system used for the detection of the probe-target hybrid, direct and indirect methods are used. 

Indirect method in which the probe-target hybrid detected by affinity cytochemistry when hapten-labeled probes are used. Different haptens such as, biotin, fluorescein and digoxigenin are used as reporter molecules and can be recognized by speciflc antibodies. 

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A diagnostic method using fluorescence labeled DNA probes to detect and quantify the number complementary chromosomal sequences on a cellular resolution. A related technique that also allows assessment of gene amplifications, but without precise quantification of copy numbers is the chromogenic in situ hybridization (CISH). Here, instead of a fluorescent dye an enzyme that can generate a colored precipitate in the tissue samples is coupled to the DNA probe. 

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]. 

Hongmanee P., Stender H., Rasmussen O.F. Evaluation of a fluorescence in situ hybridization assay for differentiation between tuberculous and nontuberculous Mycobacterium species in smears of Low-enstein-Jensen and mycobacteria growth indicator tube cultures using peptide nucleic acid probes. J. Clin. Microbiol. 2001 39 1032-1035. 

Drobniewski E.A., More P.G., Harris G. S. Differentiation of Mycobacterium tuberculosis complex and nontuberculous mycobacterial liquid cultures by using peptide nucleic acid-fluorescence in situ hybridization probes./. Clin. Microbiol. 2000 38 444-447. 

Perry-O Keeee H., Stender H., Broo-MER A., Oliveira K., Coull J., Hyldig-Nielsen J.J. Filter-based PNA in situ hybridization for rapid detection, identification and enumeration of specific microorganisms. /. Appl. Microbiol. 2001 90 180-189. 

Fluorescence in situ hybridization Permits localization of a gene to one chromosomal band. 

FISH. Fluorescent in-situ hybridization a method utilizing fluorescently labeled DNA probes to detect or confirm gene or chromosome abnormalities that are generally beyond the resolution of routine cytogenetics. 

Takahashi K, Wesselingh SL, Griffin DE, McArthur JC, Johnson RT, Glass JD (1996) Localization of HlV-1 in human brain using polymerase chain reaction/in situ hybridization and immuno-cytochemistry. Ann Neurol 39(6) 705-711... 

McManus C, Berman JW, Brett EM, Staunton H, FarreU M, Brosnan CE (1998) MCP-1, MCP-2 and MCP-3 expression in multiple sclerosis lesions an immunohistochemical and in situ hybridization study. J Neuroimmunol 86 20-29... 

Fig. 9.4 Upregulation of MCPl and CCR2 expression in neurons of the dorsal root ganglia in association with the development of neuropathic pain. Upper panels illustrate expression of CCR2 receptors by in situ hybridization in naive rats (a) and animals subjected to Chronic Compression of the DRG (CCD) a model for neuropathic pain (b). CCR2 is expressed in small sateUite cells and many neurons (white arrows). Bottom panels (c, d) illustrate the expression of MCP-1 (immu-nohistochemistry, red) under the same circumstances. Many neurons (red arrows) express the chemokine (From White et al. 2005)...

Saura, J, Bleuel, Z, Ulrich, J, Mendelowitsch, A, Chen, K, Shih, JC, Malherbe, P, Da Prada, M and Richards, JG (1996) Molecular neuroanatomy of human monoamine oxidases A and B revealed by quantitative enz5mie radioautoradiography and in situ hybridization histochemistry. Neurosci. 70 755-774. 

Kishimoto J., Keveme E., Hardwick J. and Emson P. (1993). Localization of nitric oxide synthase in the mouse olfactory and vomeronasal system — a histochemical, immunological and in-situ hybridization study. Europ J Neurosci 5, 1684-1694. 

Strotmann J., Wanner I., Helfrich T. and Breer H. (1995). Receptor expression in olfactory neurons during rat development in situ hybridization studies. Eur J Neurosci 7, 492-500. 

Zhang K, Rekhter MD, Gordon D, Phan SH. Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohisto-chemical and in situ hybridization study. Am J Pathol 1994 145(1) 114-125. 

F15. Furuta, H., Nishi, S., Le Beau, M. M., Femald, A. A., Yano, H., and Bell, G. I Sequence of human hexokinase HI cDNA and assignment of the human hexokinase m gene (HK3) to chromosome band 5q35.2 by fluorescence in situ hybridization. Genomics 36,206-209 (1996). 

With regard to the potential function of Raf-1 in mammalian germ cells, the testis is a site of abundant expression of c-ra/-l mRNAs. Northern blot and in situ hybridization analysis has shown that although c-ra/-l is expressed most abundantly in early pachytene spermatocytes, some transcripts were also detected in germ cells from type A and B spermatogonia through to the round spermatid stage (Wolfes et al., 1989 Wadewitz et al., 1993). 

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