For the ultrastructural characterization, hippocampal neurons were treated as previously described.
56 (
link) Briefly, neurons were fixed with an aldehydic solution (1.5% glutaraldehyde in 0.1 M Cacodylate buffer - pH 7.4), washed, and post-fixed with reduced osmium tetroxide solution (1% K
3Fe(CN)
6 + 1% OsO
4 in 0.1 M Cacodylate buffer). After rinses, neurons were stained with our homemade staining solution (X solution diluted 1:10 (v/v) in 20% ethanol/water,
109 (
link) then dehydrated, with an increasing series of ethanol concentrations. Samples were then embedded in epoxy resin (
Epoxy embedding medium kit, Merck KGaA, Darmstadt, Germany) which was then baked for 48 h at 60°C. After the coverslips have been removed, embedded neurons were mounted on a resin support and sectioned with a
UC7 ultramicrotome (Leica Microsystems, Vienna, Austria) with a 35° diamond knife (Diatome Ltd, Switzerlands). Sections of 80 nm were collected on 300 mesh copper grids (
G300Cu - Electron Microscopy Science, Hatfield, PA, USA).
Grids were analyzed with a Zeiss
Libra 120 Plus transmission electron microscope, operating at 120 kV, and equipped with an in-column omega filter (for the energy filtered imaging).
Falconieri A., De Vincentiis S., Cappello V., Convertino D., Das R., Ghignoli S., Figoli S., Luin S., Català-Castro F., Marchetti L., Borello U., Krieg M, & Raffa V. (2022). Axonal plasticity in response to active forces generated through magnetic nano-pulling. Cell Reports, 42(1), 111912.
