3-dimensional fluorescence in situ hybridization (3D-FISH)

Fixation 3D-FISH was performed as per standard protocols [86]. Hybridization Chromosome painting probes (Applied Spectral Imaging (ASI), Israel) were incubated at 37 °C for 5 min, denatured at 80 °C for 5 min, quick chilled on ice for 2 min and pre-annealed at 37 °C (~ 45 min). ~ 3–4 μl probe was applied to fixed cells on an 18 X 18 mm² coverslip, co-denatured at 80 °C for 5 min, and hybridized for 48 h at 37 °C. Detection Coverslips were washed in 50% FA/2X SSC (pH 7.4) - 3 X 5 min at 45 °C, 0.1X SSC - 3 X 5 min at 60 °C, rinsed in 0.1% Tween20/4X SSC, counterstained with DAPI (3 min at RT), gently washed in 2X SSC and mounted. Imaging Image acquisition was performed on a Zeiss LSM 710 confocal microscope (Carl Zeiss, Thornwood, NJ, USA) or Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) with 63X Plan-Apochromat 1.4 NA oil immersion objective. Image acquisition: zoom factor 2.0, Z-stacked images of voxel size 0.132 μm X 0.132 μm X 0.34 μm, 512X512 pixels per frame using 8-bit pixel depth for each channel. Line averaging: 2.0 and images acquired sequentially in a three-channel mode.