β casein

EpH4 cells were similarly cultured with MM, GM, or matrigel-coated GM to form the cell aggregates. After incubation for 7 days, the cell aggregates were fixed with 4 vol% paraformaldehyde at 4 °C for 1 h and embedded in optimal cutting temperature compound (Sakura Finetek Japan Ltd, Tokyo, Japan) and frozen in liquid nitrogen. The frozen samples were sectioned using a cryotome (CM3050S, Leica Microsystems, Wetzlar, Germany) and incubated at 4 °C overnight with the following primary antibodies: β-casein (Santacruz Inc., America, 1:50) or Laminin (Abcam Inc., Cambridge, UK, 1:100). Then, secondary antibodies coupled to Alexa 488 (Molecular Probes, Invitrogen Inc., Carlsbad, America, 1:700) were incubated at 25 °C for 30 min for fluorescent viewing. Next, TO-PRO-3 (Molecular Probes, Eu-gene, USA) was added to incubate at 25 °C for 10 min for the nuclear staining of cells. The sections of 10 μm thickness were viewed in a confocal laser scanning microscope (FV1000D, Olympus Ltd, Tokyo, Japan).