Vysis cep 7 d7z1 spectrumorange probe

Cells were grown on a glass coverslip and hypotonically swollen in PBS diluted to 20% with tap water for 5 min. Cells were fixed with methanol–acetic acid (3:1) for 5 min and then dried. The coverslip was hardened for 2 h at 70°C. Fixed cells were denatured with 70% formamide in 2×SSC (300 mM NaCl and 30 mM C₆H₅Na₃O₇-2H₂O, pH 7.0) for 2 min at 70°C, washed with ethanol, and dried. Denatured cells were incubated with predenatured FISH probes for centromeres of chromosome 7 and 12 (Vysis CEP 7 [D7Z1] SpectrumOrange probe and Vysis CEP 12 [D12Z3] SpectrumGreen probe, respectively; Abbott) in CEP Hybridization Buffer (Abbott) for 12 h at 37°C and washed with 50% formamide in 2×SSC and 1×SSC with DAPI. After final washes, cells were mounted with ProLong Gold (Thermo Fisher Scientific). Z-image stacks were captured in 0.2-µm increments on an Olympus IX-71 inverted microscope controlled by DeltaVision softWoRx using ×20 0.75 NA UPLSAPO objective lens (Olympus) with a CoolSnapHQ2 charge-coupled device camera. Image stacks were projected and saved as TIFF files. Fluorescence foci on the DAPI signal were counted using Speckle Inspector of BioVoxxel ToolBox plug-in (http://www.biovoxxel.de/) for Fiji (Schindelin et al., 2012 (link)). All cells analyzed were selected from nonoverlapping fields. Each experiment was successfully repeated at least three times.