Morphologic pattern of LNShh human prostate cancer cells and MC3T3 mouse pre-osteoblasts in mixed culture. MC3T3 cells were mixed with Shh-expressing LNShh cells then seeded onto chamber slides and maintained for 14 days. (Panels a, b and c) GFP-expressing LNShh cells (stably transfected with pIRES2-hShh-EGFP vector) formed clusters surrounded by a stroma of MC3T3 cells which expressed intense phalloidin staining. DAPI was used as counterstain. Cells were visualized by fluorescent microscopy. (Panel d) LNShh cells in mixed culture were further identified by positive immunocytochemical staining for human cytokeratin 8 which was not detected in surrounding MC3T3 cells. Hematoxylin was used as counterstain. Scale bars: 50 μm.