Tecnai 12 biotwin microscope

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.

For electron microscopy studies, nerve explants (not de-sheathed) at each time point were pinned to a plastic strip with sterile needles and fixed by immersing in a 2% (w/v) PFA and 2% (v/v) glutaraldehyde solution for 24 h, transferred into PBS, and refrigerated until all time points were collected. The samples were then processed by University of Manchester Electron Microscopy Facility as ultrathin nerve cross sections for TEM imaging. The nerve samples were fixed for an hour in 1% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer, followed by another 24 h in 1% uranyl acetate in water. The sample was then dehydrated in an ethanol series with 812 resin (TAAB laboratories), before polymerization for 24 h at 60°C. Samples were cut with the Reichert Ultracut Ultra-Microtome as ultra-thin sections. These were visualised with FEI Tecnai 12 Biotwin microscope at 100 kV accelerating voltage, and snapshots saved with Gatan Orius SC1000 CCD camera.