2.5. Immunocytochemistry
UMR-106 cells and PC3 cells were grown in DMEM/F-12 media supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin until the cells reached 50% confluency. Subsequently, the cells were added to transwell inserts with 3 µm pore size and the media was changed to serum free media. Cultures were incubated for 48 h prior to fixation with 4% paraformaldehyde. Cell membranes were permeabilized with 0.1% Triton X-100 prior to blocking in PBST + 10% fetal bovine serum and staining with 5 µg/mL of Sost antibody (AF1589, R & D Systems) in blocking buffer for 1 h. Subsequent secondary antibody staining was performed for 1 h with Alexa Fluor® 594 goat anti-Mouse Secondary Antibody followed by DAPI staining. Imaging was performed using a Leica DM50000B.