Fig. 4. Specificity of substrate accumulation. To determine whether tubules in matrigel cultures transport a variety of fluorescent substrates, matrigel cultures were initiated as described in the legend to Fig. 2. Confocal micrographs of matrigel cultures incubated for 20 hours in Phenol Red-free medium supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5x10^–8 M hydrocortisone, 10 ng/ml EGF and either 5,6-carboxyfluorescein (5 µM), Rhodamine 123 (10 µM) or BODIPY FL verapamil (5 µM), also as described in Fig. 3. (A) Cultures with 5,6-carboxyfluorescein (5,6 FAM) shown as a fluorescent image (white present in the lumen represents 5,6 FAM), and the same section shown in (B) as a bright-field image. (C) Fluorescent image of a culture incubated with BODIPY FL verapamil (represented by white, which is present intracellularly). (D) Fluorescent image of a culture incubated as described above with Rhodamine 123 (represented by white in cells, and diffuse gray in the lumen). Bar in A, 50 µm (same scales for B, C and D).