Epcam

In order to evaluate binding sites and their protein backbones cells on the cell surface of OH-1 cells, cultured OH-1-LUC/mCherry cells were detached with Cell Dissociation Buffer (Gibco, Carlsbad, US) and stained for sialyl Lewis A (abcam, dilution 1∶1000), CEA (Cell Signalling, dilution 1∶200), CD44 (AbD Serotec, dilution 1∶1000), PSGL-1 (Santa Cruz, dilution 1∶200), E- and P-selectin binding sites themselves using E- and P-selectin fusion proteins (R&D Systems, dilution for E-selectin 1∶1000, for P-selectin 1∶100), EpCAM (Dako, dilution 1∶59), Muc18 (GeneTex, 1∶1800) and NCAM (R&D Systems, dilution 1∶1000). The corresponding isotype controls (IgG1 for PSGL-1, EpCAM, Muc18, CEA, E- and P-selectin; IgG2a for CD44 and NCAM; IgM for sialyl Lewis A) were incubated in parallel. The antibody expression was determined with FACS CALIBUR flow cytometer (Becton Dickinson, Heidelberg, Germany) and analyzed with Win MDI 2.9 software.
In vivo grown OH-1 cells from a primary OH-1-Luc/mCherry tumor were investigated by FACS analysis. Cells were double labelled for anti-CEA and a human E- or P-selectin fusion protein.

Formalin-fixed, paraffin-embedded tumor and adjacent nontumor (>2 cm away from tumor) tissues were sectioned at 4-μm intervals. Immunohistochemical staining was carried out on both tumor and nontumor tissue slides. The slides were deparaffinized in xylene and rehydrated through graded alcohol. Endogeneous peroxidase was then inactivated with 3% hydrogen peroxide at room temperature for 20 minutes. Then the slides were soaked in 0.1 mol/L citrate buffer (pH 6.0) and placed in an autoclave at 121 °C for 2 minutes for antigen retrieval. After washing with PBS (pH 7.4), the sections were blocked with 1% BSA diluted in PBS at 37 °C for 30 minutes, and then incubated with primary antibodies at 4 °C overnight. Then the HRP-conjugated goat anti-mouse/rabbit antibody and DAB (DAKO, Glostrup, Denmark) were used. Finally, the sections were counterstained by hematoxylin and mounted. The following antibodies were used: HNF-1B (1:500, Sigma-Aldrich, St. Louis, MO), K7 (1:200, DAKO, Glostrup, Denmark), K19 (1:200, DAKO, Glostrup, Denmark), EpCAM (1:200, DAKO, Glostrup, Denmark), OV6 (1:40, DAKO, Glostrup, Denmark) and PCNA (1:4000, Cell Signaling Technology, MA, USA).
Double-fluorescence immunostaining of the tumor and nontumor tissue was performed with a sequential fluorescent method as described17 (link). Immunofluorescence was observed with the Olympus IX-71.

FTE cells were seeded at 1 × 10⁴/well in an 8-well chamberslide (BD Biosciences, San Jose, CA). Cells were transfected with the miRNA precursors on the second day, using siPORT NeoFX transfection agent as described above. After 4 days of incubation, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) and permeabilized with PBS containing 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO). After blocking with 10% FBS, mouse monoclonal antibodies for CA125 (Thermo Fisher Scientific, Waltham, MA) and EpCAM (Dako North America, Inc. Carpinteria, CA) were added and incubated at room temperature for 2 h, then with Alexa Fluor 647 conjugated secondary antibodies (Invitrogen, Carlsbad, CA). A mounting medium with DAPI (Vector Laboratories, Burlingame, CA) was used for counterstaining. Microscopic images were captured by a Leica DM IRE2 fluorescence microscope (Leica Microsystems, Bannockburn, IL) and analyzed by the OpenLab Cell Imaging System software (Leica Microsystems, Bannockburn, IL). Lysates of two OSE and two FTE primary cultures and OVCA420 cell line were analyzed using standard Western blotting with CA125 antibody (Abcam, Cambridge, MA) and chemiluminescent signals were revealed using WesternBright ECL kit (Advansta, Menlo Park, CA).