Fig. 3. Lumen formation by tubules. Lucifer Yellow accumulation (observed as green) in the lumen of a tubule in matrigel cultures, while DiI (observed as Yellow) acted as a membrane stain. Matrigel cultures were initiated as described in the legend to Fig. 2. Subsequently, the cultures were incubated for 20 hours in Phenol Red-free medium supplemented with 80 µM Lucifer Yellow, 1 µM DiI, 5 µg/ml bovine insulin, 5 µg/ml human transferrin, 5x10^–8 M hydrocortisone and 10 ng/ml EGF. (A) Transverse section of a 2-week-old culture. (B) Longitudinal section of another tubule. (C) Section of a 1-week-old matrigel culture. Red arrow, DiI stained plasma membrane; Green arrow, Lucifer Yellow in lumen. (D) Cross-section of a tubule formed by a 1-week-old matrigel culture was examined by TEM, showing a lumen (L) and nucleus (N). Bar in A, 50 µm (same scale for B and C); Bar in D, 10 µm.