Egfr fish probe

A fluorescence in situ hybridization (FISH) assay was performed on human and mouse glioblastoma tumor slides using an EGFR-FISH probe from Empire Genomics, as per the manufacturer’s instructions, as described previously.^{29 (link)} In brief, the probe was applied to the slides, covered with a glass coverslip, and sealed with rubber cement. The slides were denatured at 70°C using the ThermoBrite system (Abbott Laboratories, Chicago, IL, USA) and incubated at 37°C overnight. The slides were washed with sodium citrate buffer at 45°C for 1–2 min, rinsed in PBS containing 0.05% v/v Tween-20, and counterstained with DAPI. The slides were analyzed under a Nikon ECLIPSE 80i fluorescent microscope. Randomly chosen fields of at least 50 cells were quantified for EGFR and centromere 7 (CEP7) amplification. The FISH data were analyzed by manually counting cells with EGFR or CEP7 staining. An EGFR/CEP7 ratio of >2 was considered focal amplification, whereas a ratio of <2 was considered broad amplification.