Zen2011 blue software

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)}.

IF staining was performed using standard methods^{12 (link),40 (link)} to visualize the distribution of ICAM-2 in situ. Plated cells were washed and fixed with cold methanol. Nonspecific binding was blocked 10% goat serum (Sigma). The antibody for ICAM-2 was CBR IC2/2 (Santa Cruz), diluted 1:200 in 1% goat serum. The secondary antibody was Alexa Fluor® goat anti-mouse 488 (Life Tech, Grand Island, NY, USA), diluted 1:500 in PBS. Image acquisition was accomplished with a Zeiss Axio Observer Z. 1 microscope with Apotome2 structured illumination slider and Zen 2011 Blue software (Carl Zeiss Microscopy, Thornwood, NY, USA). Optimal exposure time was determined with unstained controls, and pixel saturation was avoided. Images were initially acquired in the Zeiss.czi proprietary format, each channel having a grayscale dynamic range of 14-bits, and then pseudocolored. Files were exported to 8-bit TIFF for contrast/brightness adjustment. Scale bars were added by Zen Blue.

The filtered and immunostained CTCs were further subtyped using additional immunomarkers. CTCs x/y placement on the filter was marked on the filter substrate and the cell placement was recorded using Zen2011 Blue software (Carl Zeiss). Samples were then archived and placed in storage at 4°C for ~2 years. Samples were removed from storage and PE fluorescence was photobleached by exposure to the excitation fluorescence (565 nm) for ~10 seconds. Samples were demounted and placed into a filter holder. Cells on filters were again permeabilized for 20 min at RT and restained using an antibody panel of CXCR4 and Vimentin, in the PE channel and eflour660 channel, respectively. Filters were washed, placed onto a microscope slide with Fluoromount-G/DAPI (Southern Biotech) and sealed with a glass cover slip. An Olympus BX54WI Fluorescent microscope with Carl Zeiss AxioCam was used to re-image all bleached CTC. Exposures were preset as 500 msec (efluor 660) and 2 sec (PE), and 10–50 msec (DAPI) for equal signal comparisons between cells. A Zen2011 Blue (Carl Zeiss) was used to process the images.