5% uranyl acetate in methanol at −90°C in a Leica AFS freeze-substitution unit, infiltrated at −45°C with Lowicryl HM-20 resin (Lowi, Waldkraiburg, MK-2206 solubility dmso Germany) and polymerized with UV light. After etching with saturated sodium ethanolate solution for 3 s, ultra-thin sections on nickel grids were treated successively with 1% human serum albumin (Wako, Osaka, Japan) with 0.1% Tween 20 in Tris-buffered saline (HTBST; pH 7.5) for 1 h, primary antibodies to GluA subunits (15 μg/ml for each) in HTBST overnight, and colloidal gold (10 nm)-conjugated antirabbit IgG (1:100; British Bio Cell International, Cardiff, UK) in HTBST for 2 h. Finally, grids were stained with uranyl acetate for 15 min.
Electron micrographs were taken with an H7100 electron microscope (Hitachi, Tokyo, Japan). For quantitative analysis, the number of metal particles and the length of synaptic membrane were measured on electron micrographs, using IPLab software (Scanalytics, Fairfax, VA, PD0332991 in vitro USA). Procedures for FISH have been reported previously (Yamasaki et al., 2010). Briefly, fresh frozen sections were hybridized with mixtures of digoxigenin (DIG)- or fluorescein-labeled cRNA probes for mouse γ-7 (nucleotide residues 181–828, AF361349.1) and 67-kDa glutamic acid decarboxylase
(GAD67; 1036–2015, NCBI Reference Sequence NM_008077) or GLAST (1571–2473, AF330257.1). Supporting Fig. S2A–C shows overall patterns of FISH labeling, which were consistent with those of in situ hybridization using radiolabeled probes (Shibata et al., 1996; Fukaya et al., 2005; Uchigashima et al., 2007). DIG and fluorescein were detected using the two-step method: the first detection with peroxidase-conjugated antifluorescein antibody (Roche Diagnostics, 1:500) for 1 h and the FITC-TSA plus amplification kit (PerkinElmer), and the second detection with
peroxidase-conjugated anti-DIG antibody (Roche Diagnostics, 1:500) for 1 h and the Cy3-TSA plus Baricitinib amplification kit (PerkinElmer). Residual activities of peroxidase introduced in the first detection were inactivated by incubation of sections with 0.6% H2O2 for 30 min. TOTO3 (Invitrogen) was used for fluorescent nuclear counterstaining. Animals were anesthetized with carbon dioxide and parasagittal cerebellar slices (250 μm thickness) were prepared from mice aged postnatal day (P)24 to P95 as described previously (Edwards et al., 1989; Hashimoto & Kano, 2003). Whole-cell recordings were made from visually identified Purkinje cell somata using an upright microscope (BX50WI; Olympus, Tokyo, Japan). Ionic currents were recorded with an Axopatch 1D (Molecular Devises, Sunnyvale, CA, USA) patch-clamp amplifier. Resistances of patch pipettes were 2–3 MΩ when filled with an intracellular solution composed of (in mm): CsCl, 60; Cs D-gluconate, 10; TEA-Cl, 20; BAPTA, 20; MgCl2, 4; ATP, 4; and HEPES, 30 (pH 7.3, adjusted with CsOH). The pipette access resistance was compensated by 70–80%. The holding potential was corrected for liquid-junction potential.