REL 2022-09-26 2022-12-13 2022-12-13 0000-0002-4814-7517 Anna Maria Steyer European Molecular Biology Laboratory Meyerhofstrasse 1 Heidelberg Baden-Württemberg Germany 69117 0000-0002-7710-5738 Hauke Werner Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany Hermann Rein Str. 3 Göttingen Lower Saxony Germany 37075 Steyer AM Möbius W 28.2 10.6019/EMPIAR-11240 FIB-SEM tissue Steyer AM Buscham TJ Lorenz C Hümmert S Eichel-Vogel MA Schadt LC Edgar JM Köster S Möbius W Nave KA Werner HB Glia Glia 2022 English 10.1002/glia.24290 36354016 EMPIAR-11214 EMPIAR-11215 EMPIAR-11216 EMPIAR-11219 EMPIAR-11220 EMPIAR-11237 EMPIAR-11238 EMPIAR-11239 FIB-SEM of mouse optic nerve of a control (CTRL, 5270) /data micrographs - single frame TIFF TIFF 1186 1 UNSIGNED BYTE 5208 50.0 4908 50.0

Optic nerve of wild type (littermates) mice sacrificed by cervical dislocation at postnatal day 75 (P75). The optic nerves were dissected and immersion fixed and prepared using the reduced osmium tetroxide – thiocarbohydrazide - osmium (OTO) method as previously described in detail (Weil et al. 2018; Steyer et al. 2020) upon implementing modifications to the original protocol first introduced by (Deerinck et al. 2010). The micrographs are obtained by FIB-SEM (Zeiss Crossbeam 540): SEM: 1.5 kV (1000 pA), ESB grid 450 V, pixel size x/y 5 nm, continuous mill-and-acquire mode; FIB: 700 pA , z-step 25 nm. Postprocessing: alignment with Fiji/ImageJ SIFT, cropped, inverted and blurred (Gaussian blur, sigma 0.5), local contrast enhancement (127, 256, 1.25).