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    Chapter14- Dual Color CISH and FISH to CISH Conversion

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    Jja

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    © All Rights Reserved
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    15 views4 pages

    Chapter14 - Dual Color CISH and FISH To CISH Conversion

    Uploaded by

    Jorge Shalo Ulloa
    Jja

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
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    Chapter 14 | Dual Color CISH and FISH to CISH Conversion Ulla Henriksen PhD, Sven Maller PAD and Andreas Schonau MS, EBA Fluorescence in stu hybridization (FISH) isa sansive yet rallablo, technique for evaluating gene status on a cell-to-cell basis. The technique has been utilized as a research tool for more than 20 yea to evaluate the presence or absence of speciic DNA sequences on ‘chromosomes (1). The method has become popular as a clagnostic {oo in clinical oncology. In tho eld of pathology, FISH has boon ‘applied as an alternative or adjunct to IHC tasting, and the method has ‘been shown to have a prognostic and a predictive value with regard to certain cancer markers 2,8). Recently, anew ISH technique based ‘on chromogenic signals instead of fuorescont signals has evolved. “The technique, chromogenic in stu hybridization (CISH), has proven ‘qualities with respect to ease of use that might explain the recent growth in popularity: 1. CISH results are easly intarpreted bythe use ofa bright fld ‘eroscope which is generally used in elagnosticaboratres. 2. CISH enables viualizaton ofthe nucious and i also able to istngulsh Invasive from in stu carcinomas. 8. CISH signals do not general fade over tm allowing the tssue ‘samples tobe archived and reviowod itr. 4. CISH resembles IHC to a largo extent (as opposed to FISH) due to the use of conventional counterstains, e.g hematoxylin, for Visualization of issue morphology Because CISH combines the genstic information from FISH with the Visualization and interpretation resembling IHC, the CISH technique {s a practical and user frendly alternative to FISH. Chromogenic In Situ Hybridization ‘The frst description ofthe C1SH procedure asa practical atsmative to FISH for the detection of genetic alterations was published in 2000 (4). Since then, many publications comparing FISH and CISH have emerged and all have reached the conclusion that CISH ls an ‘accurate, reproducibie technique with high concordance to FISH (6-9). ‘The technique is as senstive as FISH with reference to borderine and 'ow ampitication samps (9). Chromogenic visualization (colorimetric ‘mathod) is based on enzyme-conjugated antibodies thet recognize {the target of interest. Reaction of substrates with onzymas euch as horseradish peroxidase (HAP) andlor akaline phosphatase (AP) leads to.chromogen precipitates, which then can be dotectod with a bright- field microscope, Another approach for brightfeld evaluation of the ‘colorimetric method Is metallographic in stu hybridization by which «targets visualized using a probe or antibody that deposits matal ‘solectively at its binding site. One metallographic method includes sliverenhanced in situ hybxcization (or ISH). SISH is based on the ‘same basic principle as CISH, but with SISH the signal appears asa black coloration due to siver precipitation (1,11). Athough both CISH ‘and SISH have their advantages, they sufer from one drawback. In the tradtonal CISH/S'SH procedures described so fr, only signals cf one color can be detected on each side. As a consequence, the target gone and tho roferance signals are detected on two separate slides, which sometimes can be cumbersome, DuoCiSH™, a dua calor CCISH kit dovolopod by Dako Denmark A/S, overcomes this barier by simpliying the interpretation of cases with bordertine gene copy ‘number and making it sasir to visualize two probes simultancously {.g.theabiltyoditinguish between rus gene ampiiction/Sletion ‘and chromosomal aneuploidy) onthe same slide. {At prasont, only few procedures enabling datetion ot argot and reference gene on the same side apart from DuoCISH™ have been Sarl uted wich oles reayauton wit orl iets Fay sine CI an aoe the FISH pce possible to redevelop an already mounled and developed FISH sido {or reinteptetation(ee0 gure 2). Dual Color CISH and FISH to CISH Conversion Applications of Dako DuoCISH™ ‘Dako DucCISH™ can be applied to samples hybridized with a Texas, FRed-labeled probe and a FITC-labeled probe, Although the kit has ‘not yet been validated against any specific probe, the kt reagents ‘can be used to visualize genetic alterations, inckiding amplification,

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