Fluorescence In Situ Hybridization (FISH)
CECs grown in chamber slides were washed once with PBS and fixed with 4% PFA in PBS for 10 min at room temperature. Once fixed, cells were washed twice with PBS and permeabilized with 70% ethanol for 15 min at room temperature. Cells were rehydrated using a 50% formamide and 2 × SSC buffer for 5 min at room temperature. Cells were incubated overnight at 37°C in hybridization solution containing 50% formamide, 2 × SSC, 10% dextran sulfate, 0.2% BSA, 1 mg/mL yeast tRNA, and 12 μg/mL of Cy3-(CAG)7 probe. Cells were washed thoroughly using 50% formamide in 2 × SSC followed by 50% formamide in 0.1 × SSC before being stained with DAPI (1:5,000 dilution; Sigma) for 2 min at room temperature. Cells were washed with PBS and coverslips were mounted onto the microscope slide using fluorescent mounting medium (Dako). Images of foci were taken using a confocal Zeiss 700 microscope and processed with the Zeiss software.