Bx54wi fluorescent microscope

Following filtration and CTC immunostaining filters/cells can be probed using HER-2/CR-17 FISH probes performed as previously described^{25 (link), 26} with PathVysion HER-2 DNA Probe Kits, supplied by Abbott Molecular Inc. The identified CTCs x/y placement on the filter was marked on the filter substrate, and cell placement was recorded using Zen2011 Blue software (Carl Zeiss). Samples were demounted in a 2 X SSC solution for 10 min and dried by air. The protease solution was added to each sample for 20 min in a 37°C incubator. Slides were washed twice in 2X SSC for 5 min and were dried on a 45°C warmer. Slides were placed in the denaturing solution at 72°C for 5 min and were sequentially washed with 70%, 85%, and 100% ethanol for 1 min each, then dried on a 45°C warmer, followed by the addition of 10 μl of probe to the slides, a coverslip was added, and sealed with rubber cement. The slide was incubated for 22 h in a 37°C hybridization chamber. Coverslips were then removed in post-hybridization wash buffer at room temperature, washed with post-hybridization wash buffer at 72°C for 2 min, rewashed in 2× SSC for 10 min, and dried at room temperature. Samples were mounted with Fluoromount-DAPI (Southern Biotech) and imaged on an Olympus BX54WI Fluorescent microscope with a Carl Zeiss AxioCam. Images were overlaid using Zen2011 Blue software (Carl Zeiss) as described ^{25 (link)}.