Fluorescence in situ Hybridization Analysis

Fluorescence in situ hybridization analyses were performed on chromosome preparations generated from the cell lines using the following conventional techniques: colcemid arrest, hypotonic treatment, and methanol/glacial acetic acid fixation, as described previously [34 (link)]. The following bacterial artificial chromosome (BAC) clones were used: CH82-199H02 and CH82-179B09 for the MDM2 region, CH82-213B06 and CH82-204K11 for the CDK4 region, CH82-99P23 and CH82-60O16 for the CFA 7 region (58.4 Mb to 58.9 Mb), CH82-1E17 and CH82-40I15 for the region containing GABPBP1, and USP8 and TRPM7 amplifications (https://bacpacresources.org/, accessed on 1 July 2021). These BAC clones were labeled using green-dUTP (Abbott Molecular, Des Plaines, IL, USA) and Cy3-dCTP (Amersham Biosciences, Chalfont, UK). The slides were analyzed by an experienced cytogeneticist (FC) using a fluorescence microscope (Axioskop2, Axio Imager Z2, Zeiss, Göttingen, Germany) and Isis imaging software (Metasystems, Altlussheim, Germany). At least 100 non-overlapping tumor nuclei were examined in this study.

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Prouteau A., Mottier S., Primot A., Cadieu E., Bachelot L., Botherel N., Cabillic F., Houel A., Cornevin L., Kergal C., Corre S., Abadie J., Hitte C., Gilot D., Lindblad-Toh K., André C., Derrien T, & Hedan B. (2022). Canine Oral Melanoma Genomic and Transcriptomic Study Defines Two Molecular Subgroups with Different Therapeutical Targets. Cancers, 14(2), 276.

Publication 2022

green-dUTP Cy3-dCTP Axioskop2 Axio Imager Z2 Isis imaging software

Bacterial artificial chromosome Cell lines Chromosome Clones Colcemid Dctp Dutp Fluorescence in situ hybridization Fluorescence microscope Glacial acetic acid Mdm2 Methanol Nuclei Trpm7 Tumor Usp8

Corresponding Organization :

Other organizations : Institut de génétique et de développement de Rennes, Hôpital Pontchaillou, Oniris, Uppsala University, Broad Institute

Top 5 similar protocols

Variable analysis

independent variables

Fluorescence in situ hybridization
Bacterial artificial chromosome (BAC) clones used for targeting specific regions

dependent variables

Chromosome preparations generated from cell lines
Hybridization patterns observed in tumor nuclei

control variables

Colcemid arrest
Hypotonic treatment
Methanol/glacial acetic acid fixation
Fluorescence microscope used for analysis
Isis imaging software used for analysis

controls

No positive or negative controls were explicitly mentioned in the input text.

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