Glutaraldehyde

Samples were diluted to 100 μg/ml in 20 mM HEPES pH 7.4, 150 mM NaCl, 5% glycerol, 7.5 mM glutaraldehyde (Electron Microscopy Sciences) and incubated for 5 min before quenching the glutaraldehyde by the addition of 1 M Tris (to a final concentration of 75 mM) and 5 min incubation. A 5-μl drop of sample was applied to a glow-discharged carbon-coated grid (Electron Microscopy Sciences, CF300-Cu) for 10 to 15 s, blotted, stained with 2% uranyl formate (Electron Microscopy Sciences), blotted, and air-dried. Images were obtained using a Philips EM420 electron microscope at 120 kV, 82,000× magnification, and a 4.02 Å pixel size. RELION (57 (link)) software was used for particle picking and 2D and 3D class averaging.

12-week-old mice were deeply anaesthetized with ketamine hydrochloride (150 mg/kg) and medetomidine (2 mg/kg) followed by intracardiac perfusion with PBS 0.1 M followed by 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and 4% PFA in PBS 0.1 M. Brains were removed, post-fixed overnight in 2.5% glutaraldehyde and 4% PFA in PBS 0.1 M and washed 3–4 times for 1 h at 4 °C. A ~ 1 mm³ block containing the hippocampus was micro-dissected out from each whole brain before being treated with osmium tetroxide (1% in 0.1 M PBS) for 30 min. Samples were dehydrated through an ascending series of ethanol and propylene oxide, before embedding in Durcupan resin. During dehydration, sections were treated with uranyl acetate (1% in 70% ethanol) for 40 min. Durcupan resin was polymerised for 48 h in an oven set to 56 °C. Smaller regions of interest containing the CA3 region of hippocampus were identified and then cut from Durcupan-embedded sections before being mounted on Durcupan blocks and cutting ultra-thin sections (~ 70 nm) on an ultracut microtome (Leica) with an Ultra 45 Diamond Knife (Diatome, Hatfield, PA, USA). Sections were collected on formvar-coated slot grids and stained with lead citrate before imaging on a JEOL transmission electron microscope equipped with a Gatan digital camera (Jeol UK Ltd., Welwyn Garden City, UK).