Epon embedded

Anesthetized mice were perfused intracardially with 15 mL HBSS, followed by 50 mL fixative (2.5% glutaraldehyde, 4% paraformaldehyde in phosphate buffer with 0.5% NaCl). Brains were dissected and 200 μm sagittal sections cut using Leica‐VT1200S Vibratom (Leica‐Microsystems). The genu of the corpus callosum was punched out of the sliced tissue, post‐fixed with 2% OsO₄ (Science Services), dehydrated with ethanol/propylene oxide and Epon‐embedded (Serva). Semi‐thin sections were stained with methylene blue and AzurII. Ultrathin sections were contrasted with 4% uranyl acetate and lead citrate, scanned via Zeiss EM900 (Zeiss) and digital pictures obtained by wide‐angle dual speed 2K‐CCD‐Camera (TRS). Myelin/axon tracings were performed on EM pictures (≥15/animal) via ImageJ (http://rsb.info.nih.gov/ij/). As myelin thickness index and inner tongue ratio, g‐ratio and t‐ratio were calculated.

The animals used for the transmission electron microscopy (EM) experiment were perfused directly after the experiment with 5 ml of HBSS, followed by 30 ml of fixative (2.5% glutaraldehyde, 4% PFA in phosphate buffer; 0.1 M). The spinal cord at the laminectomy site was immersed in the same solution during the perfusion. After overnight fixation, the spinal cord was extracted and further post-fixed overnight at 4°C in the same fixative. The area below the laminectomy was collected and post-fixed with 2% OsO₄ and 1.5% ferricyanide (Science Services), dehydrated by ethanol, then acetone and finally Epon-embedded (Serva). Then 50-nm ultrathin sections from the area corresponding to the imaging site in the two-photon acute imaging experiment were contrasted with 4% uranyl acetate (Science Services) and lead citrate (Sigma). The imaging was done on a transmission electron microscope JEM 1400 plus (JEOL) equipped with an 8 megapixel camera (Ruby, JEOL). Large areas covering the first 20–30 µm from the dorsal pial surface of the spinal cord were imaged using the Shuttle-and-Find software at ×8000. Images were processed using the open-source image analysis software, Fiji.