Ultracut ultramicrotome

MEF cells were grown on 10 cm dishes until they reached confluency. The samples were fixed by adding double-concentrated fixative directly to the same amount of medium in the culture dish, so that the final fixative was 4% formaldehyde with 2.5% glutaraldehyde in 0.1 M HEPES buffer (pH 7.2). Then, samples were post-fixed with reduced osmium (1% osmium tetroxide with 1.5% potassium ferrocyanide) in 0.1 M cacodylate buffer (pH 7.2) for 1 h, then in 1% uranyl acetate in water overnight. The samples were dehydrated in ethanol series infiltrated with TAAB LV resin and polymerized for 24 h at 60°C. 80-nm sections were cut with a Reichert Ultracut ultramicrotome and observed with a FEI Tecnai 12 Biotwin microscope at 100 kV accelerating voltage. Images were taken with a Gatan Orius SC1000 CCD camera.

Whole-mount primary antibody labeling was performed as described above. After overnight incubation at 4°C with a rabbit pAb anti-laminin antibody (ab11575, Abcam), organoids were washed with PBS-Triton and incubated overnight at 4°C with a goat anti-rabbit IgG labeled with 10 nm gold (ab39601, Abcam) diluted 1:400. Samples were then fixed with 4% paraformaldehyde and 2.5% (wt/vol) glutaraldehyde (Agar Scientific, UK) in 0.1 M HEPES (H0887, Sigma-Aldrich) pH 7.2, and postfixed with 1% osmium tetroxide (R1024, Agar Scientific) and 1.5% potassium ferrocyanide (214022, The British Drug House, Laboratory Chemicals Division) in 0.1 M cacodylate buffer (R1102, Agar Scientific) pH 7.2 for 1 hr, then with 1% uranyl acetate (R1100A, Agar Scientific) in water for overnight. Samples were dehydrated, embedded with low-viscosity medium-grade resin (T262, TAAB Laboratories Equipment Ltd) and polymerized for 24 hr at 60°C. For transmission electron microscopy, sections were cut with a Reichert Ultracut ultramicrotome and observed with a FEI Tecnai 12 Biotwin microscope at 80 kV accelerating voltage equipped with a Gatan Orius SC1000 CCD camera.

Cal27 and 2B1 cells were grown on Aclar film (Agar Scientific Ltd.) for 7 days in culture medium and fixed with 4% (v/v) formaldehyde plus 2.5% (v/v) glutaraldehyde in 0.1 M HEPES buffer (pH 7.2). Subsequently samples were post-fixed with 1% (w/v) osmium tetroxide and 1.5% (w/v) potassium ferricyanide in 0.1 M cacodylate buffer (pH 7.2) for 1 h, then 1% (w/v) tannic acid in 0.1 M cacodylate buffer (pH 7.2) for 1 h and finally in 1% (w/v) uranyl acetate in distilled water for 1 h. Samples were then dehydrated in an ethanol series infiltrated with TAAB Low Viscosity resin and polymerized for 24 h at 60°C as thin layers on Alcar sheets. After polymerization Aclar sheets were peeled off and layers of polymerized resin with cells were re-embedded with the same resin as stacks. Sections were cut with a Reichert Ultracut ultramicrotome and observed with a FEI Tecnai 12 Biotwin microscope at 100kV accelerating voltage. Images were taken with a Gatan Orius SC1000 CCD camera.