Lsr egfr spectrum orange cep 7 spectrum green probe

Cells obtained from the first short-term culture passages were exposed to colcemid (0.02 µg/mL) for 2 h at 37 °C and harvested routinely. Metaphasic chromosomes were G-banded using a conventional trypsin-Giemsa technique and karyotypic analyses were performed according to the 2020 International System for Cytogenetic Nomenclature [54 ].
To evaluate the EGFR status, dual color FISH analysis was performed on cultured cells using the LSR EGFR Spectrum Orange/CEP-7 Spectrum Green Probe from Vysis (Abbott Laboratories, Downers Grove, IL, USA; Cat. No. 32-191053). Hybridizations were performed according to the manufacturer’s instructions and the nuclei were counterstained with DAPI. Fluorescent signals were detected using a Leica DM400B photomicroscope (Leica, Madrid, Spain) equipped with an appropriate filter set. Signals were counted in 100–150 cell nuclei. In each case the mean signal numbers for EGFR and the control CEP-7 probe were calculated and used to determine the EGFR/CEP-7 ratio. EGFR was amplified when the EGFR/CEP-7 signal ratio was > 2 [55 (link)].

GBM samples used for FISH analysis were studied using tissue microarrays (TMA). We removed four 0.6-mm cores, from paraffin blocks of each case, corresponding to tumoral areas, confirmed by hematoxylin-eosin staining, using the Beecher Instruments Manual Tissue ArrayerI (Beecher Instruments, Sun Prairie, WI, USA). FISH was carried out using the LSR EGFR Spectrum Orange/CEP 7 Spectrum Green Probe from Vysis (Abbott Laboratories, Downers Grove, IL, USA. Cat. No. 32-191053). Paraffin embedded TMAs were cut into 5-µm sections and these were mounted on Superfrost/Plus microscope slides (Microm International). Hybridizations were performed according to the manufacturer’s instructions. Counterstaining of nuclei was carried out using DAPI.
The fluorescent signal was detected using a photomicroscope, Leica DM400B, equipped with a set of the appropriate filters. Signals were counted in 100–150 non-overlapping tumor cell nuclei in the paraffin sections. The mean signal number for the EGFR gene and CEP 7 was calculated for each case, followed by the calculation of EGFR/CEP 7 ratio. The EGFR gene was quantified as amplified in individual cells when the EGFR/control signal ratio was greater than 2 [9] (link).