Ultrastructural Analysis of Neuromuscular Junction

Astrocyte, motor neuron and NMJ morphologies were evaluated with SEM at day 28 of the coculture timeline using a previously published method [25 (link), 26 ]. In brief, cultures were fixed in 2.5% glutaraldehyde (Agar Scientific, Essex, UK, Cat N° R1020) in 0.1 M Na-cacodylate buffer (Sigma-Aldrich, Cat N° C0250) and the SND75 devices were carefully removed from the Aclar sheets. Next, the cultures were incubated in 1% osmium tetroxide (Electron Microscopy Sciences, Cat N° 19150) and dehydrated in a graded ethanol series to 100% ethanol. The sheets were then critical point dried, mounted on SEM support stubs and coated with 5 nm Chromium in a Leica ACE600. Cultures were imaged with a Zeiss Sigma SEM.