Mice were injected with 10% chloral hydrate and transcardially perfused with 0.1 M sodium cacodylate buffer followed by ice-cold 2% PFA and post-fixed with 0.5% PFA at 4 °C. Tissue was sectioned on a vibratome, and freeze substitution and low temperature embedding of the specimens was performed as described previously83 (link),84 (link),85 (link). Slices were cryoprotected by immersion in increasing concentrations of glycerol (from 10% to 30% in PBS) (v/v). Sections were plunged rapidly into liquid propane cooled by liquid nitrogen (−190 °C) in a Universal Cryofixation System KF80 (Reichert-Jung, Vienna, Austria). The samples were immersed in 1.5% uranyl acetate dissolved in anhydrous methanol (−90 °C, 24 h) in a cryosubstitution AFS unit (Leica, Vienna, Austria). The temperature was raised from −90 °C to −45 °C in steps of 4 °C/h. After washing with anhydrous methanol, the samples were infiltrated with Lowicryl HM20 resin (Electron Microscopy Sciences, Fort Washington, PA) at −45 °C. Polymerization with ultraviolet light (360 nm) was performed for 48 h at −45 °C, followed by 24 h at 0 °C. Ultrathin sections (80 nm) were cut with a diamond knife on a Leica UC7 ultramicrotome and mounted on 300 mesh copper grids using a Coat-Quick adhesive pen (Electron Microscopy Sciences). Images (n=10/animal) were taken using a Hitachi 7700 electron microscope (Hitachi High-Technologies Corporation America, Inc.) equipped with a XR81-B-M1-BT-FX, 8 Megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA). Images were then imported into Adobe Photoshop (Adobe, 2022) and the extracellular space was manually scored using a computer tablet. Scoring was done by two independent investigators blinded to experimental conditions. Images were then imported into ImageJ (v1.53f51)86 (link) and the percentage of marked area/total area was calculated.
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