Fluorescence in-situ hybridization method

Radiation Personnel Accident. Radiation exposure in accidents of personnel in nuclear facilities and non-destructive detection have been evaluated with ESR dosimetry. For example, the amputated medium fibre bone of a worker in Peru, who tried to solve the problem by directly touching the source of 60Co unit for a few seconds, was measured four years later.The dose was measured by 6.4 + 0.5 Gy, while 20 + 3 Gy was obtained from fluorescence in situ hybridization method. The result would suggest diagenic decay of the signal due to the metabolism of bone.96 Thus, ESR gives the retrospective dose for radiation accidents. 

Brockman SR, Paternoster SR Ketterling RR Dewald GW. New highly sensitive fluorescence in situ hybridization method to detect PML/RARA fusion in acute promyelocytic leukemia. Cancer Genet Cytogenet 2003 145 144-151. 

Ahneida, C., Azevedo, N. R, Fernandes, R. M., Keevil, C. W., Vieira, M. J. (2010). Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples. Applied and Environmental Microbiology, 76(13), 4476-4485. http //dx.doi.org/10.1128/AEM.01678-09 PMCID PMC2897454. 

Beimfohr, C. Thelen, K. Snaidr, J. A fluorescence in situ hybridization method for detection of microorganisms using FRET probes to increase signal noise ratio. Ger. Offen. DE 102010012421, 2010. 

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. 

A variation on this method, called fluorescent in situ hybridization (FISH), uses fluorescent-labeled DNA and RNA probes for detection and visualization of single cells by microscopy or flow cytometry.7 80 The FISH technique is popular because of its sensitivity and speed of visualization fluorescent dyes can be used to produce probes with different colors for simultaneous detection of several organisms.76,81,82... 

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/... 

Bouin s solution is one of the traditional ways to harden cell pellet. Some cytologists believe it provides the best cellular details, especially nuclear features in cell blocks.28 The major steps are (1) After centrifugation, fix the cell pellet with Bouin s solution. (2) After 2h, discard the solution. (3) Remove the hardened cell pellet from the tube, wrap it with lens paper, and transfer it into a cassette for further processing. We have been using this method for many years. In our experience, most of the time, ICC results are consistent with IHC from the surgical specimen. The biggest drawback of this method is the toxicity of Bouin s fixative which creates biohazard and safety issues for the laboratory. We also found cell blocks gave poor fluorescence in situ hybridization (FISH) results after Bouin s fixation. 

Meyne, J. (1993) Chromosome mapping by fluorescent in situ hybridization. In Methods in Nonradioactive Detection (G.C. Howard, ed.), pp. 263-268. Appleton Lange, Norwalk, Connecticut. 

Method/technique Fluorescent in situ hybridization (FISH)... 

The RNA molecules, ribosomal RNA (rRNA) and messenger RNA (mRNA) play key roles in the protein synthesis. The amount of RNA in individual cells or in a community may, therefore, be taken as an indicator of protein synthesis and, thus, microbial activity. The number of active cells can be detected by fluorescent in situ hybridization (FISH) (Amann et al. 1995). By this method, individual cells carrying high concentrations of rRNA, situated on ribosomes, are quantified by fluorescence microscopy. The amount of rRNA in a community can also be detected by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), where rRNA extracted from soil is detected by creating a DNA copy and separating by gel electrophoresis (Duineveld et al. 2001). 

Finally, several PNA based methods for use in genetic diagnostics have been developed. Of particular interest are methods for fluorescence in situ hybridization (FISH), modulation of polymerase chain reaction (PCR) analyses, and detection of genetic material by sensors and mass spectrometry. 8 ... 

A number of methods are available for analyzing tumor HER-2 status. The selection of the method depends on the target molecule to be detected. The target molecules are DNA mRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot (Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al 1993), fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH) on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction (Gramlich et al., 1994). Assays to determine mRNA oveiexpiession include Northern blot (Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) and ISH (Naber et al., 1990). Methods to assess HER-2/mcm protein product overexpression... 

Pauletti, G., Dandekar, S., Rong, H.-M., Ramos, L., Peng, H., Seshadri, R., and Slamon, D. J. 2000. Assessment of methods for tissue-based detection of the HER-2/wew alteration in human breast cancer A direct comparison of fluorescence in situ hybridization and immunohistochemistry. J. Clin. Oncol. 18 3651-3661. 

Moter, A., and U. B. Gobel. 2000. Fluorescent in situ hybridization (FISH) for direct visualization of microorganisms. Journal of Microbiological Methods 41 85—112. 

Nielsen K V, Muller S, Agerholm I, Poulsen T S, Matthiesen S H, Witton C J, Schonau A. PNA suppression method combined with fluorescence in situ hybridization (FISH) technique. In PRINS and PNA Technologies in Chromosomal Investigation. Frank Pellestor, eds. NOVA Publishers,... 

The above studies established a precedent for the combined use of microbiological, genomic, and isotopic methods to study important biogeochemical processes. Eurthering the revolution of culture-independent methods for studying these processes on natural samples has been the visualization of the active microbes through fluorescence in situ hybridization (Boetius et al., 2000),... 

---