4-μm-thick slides of FFPE were used with Vysis MET SpectrumRed FISH Probe and Vysis centromere of chromosome 7 (CEP7) SpectrumGreen Probe (Abbott Molecular, Chicago, IL, USA) to investigate MET gene copy number (GCN) status according to the manufacturer's instructions. The red fluorescent probe specific for the MET gene and the green fluorescent probe as a reference locus of MET specific for the CEP7. Gene GCN was reported by two methods: mean MET copy number per cell (mean MET/cell) and the MET copy number per centromere 7 ratio (MET/CEP7) [27 (link)]. MET gene and CEP7 copy number per cell and MET/CEP7 ratio were counted in at least 50 non-overlapping tumor cell nuclei, at a magnification of 100 x. MET amplification was defined according to the previously reported study, proposed as follows: MET gene mean copy number no less than 5 and a MET/CEP7 ratio greater than 2 [28 (link)]. Otherwise, a tumor was defined as negative amplification.
Spectrumgreen probe
Manufactured by Abbott
Sourced in France
The SpectrumGreen probe is a laboratory equipment product. It is designed to perform specific functions within a laboratory setting. The core function of the SpectrumGreen probe is to provide accurate and reliable measurements, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Axioplan 2 fluorescence microscope, by Zeiss (2 mentions)
Vysis met spectrumred fish probe, by Abbott (1 mentions)
Lsi fgfr2 spectrum orange probe, by Abbott (1 mentions)
Quant it dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Rna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Vectashield antifade medium, by Vector Laboratories (1 mentions)
Isis software, by MetaSystems (1 mentions)
Vectashield antifade medium containing 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
6 protocols using spectrumgreen probe
1
MET Gene Copy Number Evaluation
2
FGFR2 Gene Amplification by FISH
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The immunohistochemistry-positive areas of tumor formalin-fixed paraffin-embedded blocks were subjected to FISH. Tumor sections were cut to 1 μm thickness, followed by deparaffinization with the pretreatment reagent (Abbott, 30-801250) at 80 °C for 30 min. Protease digestion procedures were performed using the protease reagent (Abbott, 30-801255) at 37 °C for 20 min. FGFR2 probes (LSI FGFR2 Spectrum Orange Probe, 08N42-020) and CEP 10 (Spectrum Green Probe, 06J37-020) from Vysis (Abbott Molecular, Illinois) were hybridized at 73 °C for 5 min and 37 °C for 20 h. After hybridization, the slides were washed in 2 × saline-sodium citrate/0.3% NP-40 at 72 °C for 5 min, air dried, and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) I and DAPI II (Abbott Molecular). The slides were examined under a fluorescence microscope equipped with Spectrum Texas Red with isothiocyanate and DAPI filters. The FGFR2/CEP 10 ratio were established after counting at least 50 tumor cell nuclei. An FGFR2/CEP 10 ratio higher than 2.0 was interpreted as gene amplification positive. FGFR2 gene copy numbers more than 4 without gene amplification were interpreted as FGFR2 polysomy.
3
Cytogenetic and Molecular Profiling of MDS
4
Correlating UNR with Chromosomal and Transcript Profiles
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To correlate UNR association with the chromosomal content (DNA FISH) or with the poly(A) tail of mRNA (RNA FISH), sequential fluorescent labeling was performed using 6-μm tissue sections, prepared from formalin-fixed paraffin-embedded human Hep-3B xenografts [10 (link)]. Firstly, UNR immunodetection was performed, images were captured and coordinates of each image were recorded to ensure repositioning of the slide to the same area after FISH assay. Secondly, interphase DNA or RNA FISH detection was performed using the same slide; the images were captured using the repositioning function of the microscope (Zeiss Axioplan 2 fluorescence microscope, Zeiss, Jena, Germany).
DNA FISH targeted the short arm of chromosome 6 (chromosome region 6p25) (SpectrumRed probe, Abbott France, Rungis, France) and the long arm of chromosome 11 (chromosome region 11q13.3) (SpectrumGreen probe, Abbott France) and was performed as previously described [13 (link)]. RNA FISH targeting polyadenylated mRNA was also performed as described [6 (link)]. At each step, slides were mounted with Vectashield antifade medium containing 4’,6-diamidino 2-phenylindole (DAPI) (Vector Laboratories, Laboratoires Eurobio/Abcys, Les Ulis, France).
DNA FISH targeted the short arm of chromosome 6 (chromosome region 6p25) (SpectrumRed probe, Abbott France, Rungis, France) and the long arm of chromosome 11 (chromosome region 11q13.3) (SpectrumGreen probe, Abbott France) and was performed as previously described [13 (link)]. RNA FISH targeting polyadenylated mRNA was also performed as described [6 (link)]. At each step, slides were mounted with Vectashield antifade medium containing 4’,6-diamidino 2-phenylindole (DAPI) (Vector Laboratories, Laboratoires Eurobio/Abcys, Les Ulis, France).
5
FISH Analysis of MET Locus
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The MET 7q31.2 chromosomal locus was labeled with LSI MET Spectrum Red Probe (Abbott, Abbott Park, IL, USA). Centromere 7, labeled with Spectrum Green Probe (CEP7 (D7Z1), Abbott), was paired to a control for copy number. Then FISH was carried out using standard methods.23 Only nuclei with unambiguous CEP7 signals were scored for MET signal number.
6
Fluorescence In Situ Hybridization for Xenograft Analysis
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Tissue sections (6-μm thick) were prepared from formalin-fixed paraffin-embedded xenografts and FISH experiments were conducted as previously described (Pham-Ledard et al., 2014 (link)). Dual-color hybridizations were conducted with labeled probes hybridizing to the band on the short arm of chromosome 5 (chromosome region 5p15.2) (SpectrumGreen probe, Abbott France, Rungis, France) and to the chromosome 15 centromere (SpectrumOrange probe, Abbott France) according to manufacturer's protocol. After post-hybridization washing, slides were mounted with Vectashield antifade medium containing 4′,6-diamidino 2-phenylindole (DAPI) (Vector Laboratories, Laboratoires Eurobio/Abcys, Les Ulis, France) and observed on a Zeiss Axioplan 2 fluorescence microscope (Zeiss, Jena, Germany). Images were captured with a high-resolution camera using Isis software (MetaSystems, Altlussheim, Germany) with subsequent counting of FISH signals, each count being performed on 100 nuclei per mice.
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