Lsm 5 3.2 image capture and analysis software

After CD45 depletion and 84-1 selection, cells were plated on a glass slide with Cytofuge (Iris). For intracellular EpCAM staining, cells were fixed using 4% paraformaldehyde for 15 min, washed with PBS (pH 7.4), and permeabilized in PBS (pH 7.4)/0.2% NP40 (Sigma Aldrich) for 20 min. These cells were then blocked in 10% fetal calf serum (Gibco, Invitrogen) for 1 h, and labeled with EpCAM, 84-1 or CD45 primary antibody (1:100) overnight at 4°C. Cells were then rinsed in PBS (pH 7.4) and stained with Alexa Fluor-555 secondary antibodies (Invitrogen) (1:250) for EpCAM (species: rabbit), Alexa Fluor-647 for 84-1 (species: mouse). For nuclei staining, Sytox Green (Invitrogen) (1:1000) was incorporated along with secondary antibody for 60 min. The cells were then washed with PBS (pH 7.4) three times for 15 min each and mounted in Slow fade antifade (Invitrogen). For confocal analysis, images were acquired in 8 bits with the Zeiss LSM 510 confocal microscope using LSM 5 3.2 image capture and analysis software (Zeiss). A 63× water-immersion objective lens (NA, 1.0) was used with digital zoom for image capture. All images were acquired by the same operator using the same intensity and photo detector gain in order to allow quantitative comparisons of relative levels of immunoreactivity between different samples.

For immunofluorescence, the cell pellet was mixed with MACS buffer (Miltenyi Biotec) and stained with 84-1 antibody (1:200) in tube at room temperature for 1 hour. Cells were cytospun onto Polysine^™ microscope adhension slides (Thermo Fisher Scientific) by Cytofuge (Iris, Westwood, MA). Cells were fixed with 4% paraformaldehyde (Fisher Scientific) and blocked in blocking buffer (1% FBS in PBS) for 1 hour. For the staining of other markers, such as CD45 (1:100; Abcam, Cambridge, MA), α-SMA (1:100; Abcam), CD117 (1:100; Cell Signaling Technology, Danvers, MA), and MDM2 (1:100; Santa Cruz Biotechnology Santa Cruz, CA), the cells were incubated with primary antibody overnight in a cold room followed by permeabilization in PBS (pH 7.4)/0.2% NP40 (Sigma Aldrich) for 20 minutes. Next, the slides were washed with PBS three times and stained with Alexa Fluor-555 secondary antibody (1:100; Invitrogen, Carlsbad, CA) for CD45, Alexa Fluor-647 (1:100; Invitrogen) for 84-1, and Sytox Green (1:200; Invitrogen) for nuclei staining for 1 hour at room temperature. Then the slides were washed with PBS three times and mounted in Slow fade antifade (Invitrogen). All slides were visualized by Zeiss LSM 510 confocal microscope using LSM 5 3.2 image capture and analysis software (Carl Zeiss, Thornwood, NY).