In cell analyzer 2000 cell imaging system

Immunohistochemical detection of beta-catenin, and PrKD1 was performed on mouse prostate tissue and transfected and non-transfected cell lines using anti-beta-catenin (Santa Cruz, #SC1496) and anti PKC mu (Santa Cruz, # 693)⁸. Mouse prostate tissue was fixed in 4% paraformaldehyde. The paraffin-embedded paraformaldehyde-fixed mice tissue was cut at 5 micron thickness. The first slice of staining sections was used for histological confirmation by Hematoxylin and Eosin (H&E) staining. The tissue slides and fixed cells were treated with 0.1% Triton-X-100 for 3 min, followed by blocking nonspecific binding using protein block solution (Dako, #X0909), overnight incubation with primary antibody at 4°C and one hour incubation with fluorescent secondary antibodies AF594 (Life technologies #SA5-10088) or AF488 (life technologies, #A11070) and contra-stained with DAPI for 5 minutes. Goat isotype IgG (Santa Cruz) was used as negative controls. Tissue slides were examined using Olympus Fluoview (FV10i) confocal microscope and cells were analyzed by IN Cell analyzer 2000 cell imaging system (GE Healthcare Life Sciences).

Primary GBM cells were plated at 100% cell confluence in 96-well plates (Corning) coated with 10 μg/ml laminin (Sigma). We also added a red dye (Invitrogen, CellTracker Deep Red C34565) at a concentration of 5 μM as a cell tracker. Mitomycin C (Sigma) at 10 μg/ml concentration was added to the media for 40 min to stop cell proliferation. After mitomycin incubation, new media were added, and cells were imaged every 24 h for 7 days using IN Cell Analyzer 2000 cell imaging system (GE Healthcare). Cell aggregation was calculated as area covered by cells over empty surface and measured using IN CELL developer toolbox 1.9.2 software. Experiments were performed in three biological replicates (n = 3).