R- and G-banding chromosomal analysis and fluorescence in situ hybridization (FISH) analysis followed standard procedures. Probes used for FISH analysis are listed in Table S1 . Non-commercial probes were labeled with SpectrumOrange- and SpectrumGreen-d-UTP (Abbott Molecular, Ottigne, Belgium) using random priming. FISH experiments were evaluated using an Axioplan 2 fluorescence microscope equipped with a charge-coupled device Axiophot 2 camera (Carl Zeiss Microscopy, Jena, Germany) and a MetaSystems Isis imaging system (MetaSystems, Altlussheim, Germany). Two to 10 abnormal metaphases and/or 200 interphase cells were evaluated in each FISH experiment.
Isis imaging system
Manufactured by MetaSystems
Sourced in Germany
The ISIS imaging system is a laboratory equipment product that provides high-quality imaging capabilities. It is designed to capture and analyze images for various applications in a research or scientific setting. The core function of the ISIS imaging system is to enable users to acquire, process, and manage digital images with precision and efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Spectrum orange, by Abbott (4 mentions)
Spectrum green dutp, by Abbott (4 mentions)
Axiophot 2 camera, by Zeiss (4 mentions)
Bx60 epifluorescence microscope, by MetaSystems (2 mentions)
Axiophot 2, by Zeiss (1 mentions)
Nick translation, by Roche (1 mentions)
Ikaros imaging system, by MetaSystems (1 mentions)
Igh break apart probe, by MetaSystems (1 mentions)
Dhr123, by Thermo Fisher Scientific (1 mentions)
Metafer slide scanning system, by MetaSystems (1 mentions)
Metafer4, by MetaSystems (1 mentions)
Mll bap, by Abbott (1 mentions)
Telvision 11q d11s1037, by Abbott (1 mentions)
Cep11, by Abbott (1 mentions)
Facscalibur, by BD (1 mentions)
Axioplan 2 mot, by Zeiss (1 mentions)
Axioplan 2 fluorescence microscope, by Zeiss (1 mentions)
18 protocols using isis imaging system
1
Chromosome Analysis via FISH
2
Cytogenetic Analysis using G-banding and FISH
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G-banding chromosomal analysis and fluorescence in situ hybridization (FISH) followed routine methods. Probes applied for FISH are listed in Table S1 . Non-commercial probes were labeled with SpectrumOrange- and SpectrumGreen-d-UTP (Abbott Molecular, Ottigne, Belgium) using random priming. FISH images were acquired with a fluorescence microscope equipped with an Axiophot 2 camera (Carl Zeiss Microscopy, Jena, Germany) and a MetaSystems ISIS imaging system (MetaSystems, Altlussheim, Germany). One to ten abnormal metaphases and/or 200 interphase cells were evaluated in each experiment.
3
Cytogenetic and FISH Analysis Protocol
4
Chromosomal Analysis via Giemsa and FISH
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Conventional Giemsa (G)-banding chromosomal analysis and FISH analysis followed routine protocols. The probes applied on patient material and Ba/F3 cell lines are listed in the Online Supplementary Table S1. Non-commercial probes were directly labeled with SpectrumOrange- and SpectrumGreen-dUTP (Abbott Molecular, Ottignies, Belgium) by random primed label ing. FISH images were acquired with a fluorescence microscope equipped with an Axiophot 2 camera (Carl Zeiss Microscopy, Jena, Germany) and a MetaSystems Isis imaging system (MetaSystems, Altlussheim, Germany).
5
Quantifying Copy Number Variations via POD-FISH
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POD-FISH has already been successfully used to identify CNVs in human cells [8 (link), 34 (link), 35 (link)]. BAC clones for CNV regions were purchased from the Children’s Hospital Oakland Research Institute, Oakland, CA, USA, or kindly provided by the Sanger Centre, UK. BAC DNA was isolated, PCR amplified, and labeled by Nick translation (Roche, Karlsruhe, Germany) [36 (link)]. The following BACs were used: RP11-393 N21 for 1p31.1 (TexasRed), RP11-1129E22 for 7q11.22 (SpectrumGreen), RP11-174 K23 for 9q21.3 (TexasRed), RP11-123 L21 for 10q21.1 (SpectrumOrange) and RP11-264 M12 for 16q23.1 (SpectrumGreen). The set of CNV regions was selected on the base of results of genomic distribution of low-dose ionizing radiation-induced CNVs [7 (link)]. Image capturing and acquisition were processed with the Isis imaging system (MetaSystems, GmbH, Altlussheim, Germany). For analysis of POD-FISH signals the ImageJ freeware was applied (https://imagej.nih.gov/ij/ ) [37 ]. For that purpose images were imported into ImageJ program and size of CNVs was measured on the base of fluorescence intensities of signals from 50 to 60 metaphases for each chromosome region and expressed in arbitrary units (a. u.).
6
Simultaneous Whole Chromosome Painting in Human Cells
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24-color-FISH using simultaneous all human whole chromosome painting (WCP) probes was done as previously reported[21 (link),22 ]. A total of 20 metaphases was analyzed, using a fluorescence microscope (Axio Imager Z1 mot; Carl Zeiss AG, Oberkochen, Germany) equipped with appropriate filter sets to discriminate between a maximum of five fluorochromes and the counterstain DAPI; the latter was used to induce a GTG-like banding pattern. Image capturing and processing were carried out using ISIS imaging system (MetaSystems, Altlussheim, Germany).
7
Interphase FISH Validation of aCGH
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Cell suspensions were obtained from primary cultures using standard techniques (hypotonic treatment and methanol/acetic acid fixation). Interphase FISH (iFISH) was used to confirm and validate the aCGH results. In addition, this approach was used in samples which the aCGH analyses was not possible. Dual color iFISH experiments with commercially available probes were used according to standard procedures and/or to manufacturer’s instructions: TP53/CEN 17 Dual Color Probe (ZytoVision GmbH, Germany), PTEN/CEN 10 Dual Color Probe (ZytoVision GmbH, Germany), CDKN2A/CEN 9 Dual Color Probe (ZytoVision GmbH, Germany). Cells were counterstained with DAPI (4′,6-diamidino-2-phenylindole) and the slides were analyzed using a fluorescence microscope (AxioImager.Z1 mot, Zeiss) equipped with appropriate filter sets. The images were captured by a sensitive CCD camera and the result was processed by ISIS imaging system (MetaSystems, Altlußheim, Germany). In each sample, hybridization signals of at least 200 tumor cell interphase nuclei were counted. When ≥10% of the nuclei showed a decreased number of fluorescent signals of locus specific probe with respect to normal diploid cells, it was considered a loss event. For statistical value analyses, monosomy was included as heterozygous allelic loss.
8
Cytogenetic and FISH Analysis of Bone Marrow
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For conventional cytogenetic analysis, BM specimens were cultured for 24 and 72 h without mitogens, and stained by G-banding, following the standard techniques. In most of the cases, at least 20 metaphases were analyzed using an IKAROS imaging system (Metasystems, Altlussheim, Germany).
Interphase FISH (iFISH) analysis was performed using IgH Breakapart probe (Metasystems, Altlussheim, Germany), according to the manufacturer’s instructions. A minimum of 200 interphase nuclei and 10 metaphases were analyzed using a fluorescence microscope (AxioImager.Z1 mot, Carl Zeiss Ltd., Hertfordshir, UK) with ISIS imaging system (Metasystems, Altlussheim, Germany). The karyotypes were interpreted using the International System for Human Cytogenetic Nomenclature (ISCN 2016) [28 ].
Interphase FISH (iFISH) analysis was performed using IgH Breakapart probe (Metasystems, Altlussheim, Germany), according to the manufacturer’s instructions. A minimum of 200 interphase nuclei and 10 metaphases were analyzed using a fluorescence microscope (AxioImager.Z1 mot, Carl Zeiss Ltd., Hertfordshir, UK) with ISIS imaging system (Metasystems, Altlussheim, Germany). The karyotypes were interpreted using the International System for Human Cytogenetic Nomenclature (ISCN 2016) [28 ].
9
Detecting ROS in Yeast Cells
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ROS in yeast cells were detected by using two different molecular probes; Dihydroethidium (DHE; Merck Biochemicals, Darmstadt, Germany) and Dihydrorhodamine 123 (DHR123; Thermo Fischer Scientific, Schwerte, Germany).
DHE is a free radical sensor that, in its reduced form, exhibits blue fluorescence in the cell. When oxidized largely by superoxide to ethidium, it obtains a red fluorescence. DHE was added to 1 mL sporulation medium to a concentration of 20 µM, followed by incubation for 20 min before IR or H2O2 treatment. Hoechst 33342 was added to the sporulation medium to give a final conc. of 0.5 µg/mL for staining the nucleus in live cells. Finally, cells were washed twice with sporulation medium and embedded in antifade solution containing 0.25 µm TetraSpecks (Thermo Fisher Scientific; diluted 1/1000) to normalize digital image recording. Images were taken using appropriate filter sets of a Zeiss Axioimager Z2 epifluorescence microscope and the ISIS imaging system (MetaSystems, Altlussheim, Germany). Cells that fluoresced red were scored as ROS-positive.
DHR123 is a non-fluorescent radical sensor in its reduced form. When oxidized to rhodamine, it obtains a green fluorescence. Cell loading with DHR123 was done as described for DHE staining, with the final concentration of DHR123 being 100 µM.
DHE is a free radical sensor that, in its reduced form, exhibits blue fluorescence in the cell. When oxidized largely by superoxide to ethidium, it obtains a red fluorescence. DHE was added to 1 mL sporulation medium to a concentration of 20 µM, followed by incubation for 20 min before IR or H2O2 treatment. Hoechst 33342 was added to the sporulation medium to give a final conc. of 0.5 µg/mL for staining the nucleus in live cells. Finally, cells were washed twice with sporulation medium and embedded in antifade solution containing 0.25 µm TetraSpecks (Thermo Fisher Scientific; diluted 1/1000) to normalize digital image recording. Images were taken using appropriate filter sets of a Zeiss Axioimager Z2 epifluorescence microscope and the ISIS imaging system (MetaSystems, Altlussheim, Germany). Cells that fluoresced red were scored as ROS-positive.
DHR123 is a non-fluorescent radical sensor in its reduced form. When oxidized to rhodamine, it obtains a green fluorescence. Cell loading with DHR123 was done as described for DHE staining, with the final concentration of DHR123 being 100 µM.
10
ALK FISH Rearrangement Detection Protocol
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Fluorescence in situ hybridization was performed on unstained 4 μm formalin-fixed paraffin embedded tumor tissue sections with the use of an ALK break-apart probe set (Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe; Abbott Molecular, Rungis, France) using a paraffin pretreatment reagent kit (Vysis, Abbott Molecular). Assays were performed following the manufacturer’s instructions. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole/Vectashield (Vektor Laboratories, AbCys, Paris, France).
Sections were analyzed with a Metafer slide scanning system (Metasystems, Altlussheim, Germany) under a 63 × oil immersion objective with a fluorescence microscope (M1, Zeiss, Stuttgart, Germany) equipped with appropriate filters, a charge-coupled device camera, and the FISH imaging and capturing software Metafer 4 (Metasystems). Signals were enumerated with the ISIS imaging system (Metasystems). Non-rearranged ALK showed fusion (orange signals) or very close apposition of the probes adjacent to the 3′ (red) and 5′ (green) ends of the gene. Rearranged ALK appeared as split 3′ and 5′ signals. Tumor tissues were considered ALK-FISH positive (ALK rearranged) if >15% of tumor cells showed split red and green signals and/or single red signals, according to previous publications.3 (link),7 (link),10 (link),11 (link) Otherwise the samples were considered ALK-FISH negative.
Sections were analyzed with a Metafer slide scanning system (Metasystems, Altlussheim, Germany) under a 63 × oil immersion objective with a fluorescence microscope (M1, Zeiss, Stuttgart, Germany) equipped with appropriate filters, a charge-coupled device camera, and the FISH imaging and capturing software Metafer 4 (Metasystems). Signals were enumerated with the ISIS imaging system (Metasystems). Non-rearranged ALK showed fusion (orange signals) or very close apposition of the probes adjacent to the 3′ (red) and 5′ (green) ends of the gene. Rearranged ALK appeared as split 3′ and 5′ signals. Tumor tissues were considered ALK-FISH positive (ALK rearranged) if >15% of tumor cells showed split red and green signals and/or single red signals, according to previous publications.3 (link),7 (link),10 (link),11 (link) Otherwise the samples were considered ALK-FISH negative.
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