To determine the effect of coculture on differentiation into myofibroblasts, immunocytochemistry was performed with a labeled streptavidin-biotin kit (Histostain-SP; Zymed, San Francisco, CA) for the detection of α-SMA, a marker for myofibroblasts. A volume of 1.5 mL of the remaining cell suspension was mixed with 1.5 mL of TC-199 medium containing 10% FBS. The cells were then centrifuged and resuspended in 0.4 mL fresh TC-199 medium containing 10% FBS. Dissociated cells were replated onto eight-chamber slides (Laboratory-Tex; Nunc, Naperville, IL). The slides were incubated for 12 hours to permit cell adhesion, after which the cells were rinsed three times with phosphate-buffered saline and then immersed in 95% ethanol containing 0.1% Triton-X (Wako Pure Chemical, Osaka, Japan) at 4°C for fixation. After fixation, the cells were rinsed three times with phosphate-buffered saline and were immunostained for α-SMA, according to the manufacturer’s instructions. The primary antibody used was a mouse monoclonal antibody directed against human α-SMA (IgG2a, clone 1A4, code no. M851; Dacopatts, Glostrup, Denmark). Peroxidase visualization was accomplished by adding a solution containing 3-amino-9-ethylcarbazole (AEC) and hydrogen peroxide. Finally, the cells were counterstained with hematoxylin. At least 200 cells were counted in each gel to determine the ratio of the number of positive cells to total number of cells (P/T ratio). The ratio was used to assess myofibroblastic differentiation. To determine the number of myofibroblasts per gel, we multiplied the total cell number per gel by its P/T ratio.