Lowicryl hm20 resin

Freeze substitution and low-temperature embedding of the 400-μm sections were performed as previously described (Wang et al., 2010 (link); Crimins et al., 2016 ). Briefly, after the sections were cryoprotected by immersion in increasing concentrations of glycerol in PB, they were plunged in liquid propane cooled by liquid nitrogen in a Universal Cryofixation System KF80 (ReichertJung). Then the sections were immersed in 1.5% uranyl acetate in anhydrous methanol (−90°C) for 24 hr in a cryosubstitution Automatic Freeze Substitution System (Leica) and the temperature was raised 4 °C/h from −90 to −45 °C. After washing with anhydrous methanol, the sections were infiltrated with increasing concentrations of Lowicryl HM20 resin (Electron Microscopy Sciences) for 1 hr each, followed by pure Lowicryl overnight. The resin was polymerized with UV light (360 nm) at −45 °C for 48 hr, followed by room temperature for 24 hr. Seven or more consecutive ultrathin sections were cut into 90 nm-thick sections with a Diatome diamond knife (Electron Microscopy Sciences) and mounted on each formvar-supported slot grid (Electron Microscopy Sciences).

Electron microscopy (EM) and immunogold labeling was performed as previously described [18 (link), 22 ] at the Yale School of Medicine’s Center for Cellular and Molecular Imaging. Briefly, cells were fixed by high-pressure freezing (Leica EM HPM100) and freeze substitution (Leica AFS) was carried out at 2,000 pounds/square inch in 0.1% w/v uranyl acetate/acetone. Samples were infiltrated into Lowicryl HM20 resin (Electron Microscopy Science) and sectioned onto Formvar/carbon-coated nickel grids for immunolabeling.
Samples were blocked in 1% w/v fish skin gelatin, then incubated with primary antibodies diluted 1:50 in blocking buffer. 10 nm protein A-gold particles (Utrecht Medical Center) were used to detect the primary antibodies and grids were stained with 2% w/v uranyl acetate and lead citrate. Images were captured with an FEI Tecnai Biotwin TEM at 80Kv equipped with a Morada CCD and iTEM (Olympus) software.

Tissue from a normal DOCK10-Cre mouse and two pilocarpine-treated mice with unilateral δ-eGFP subunit transfection was prepared for postembedding immunogold labeling for the δ subunit, as described previously (Zhang et al., 2007 (link)). Following perfusion of the mice with 4% paraformaldehyde and 0.1% glutaraldehyde and removal of the brain, coronal sections of the forebrain that included the hippocampus were cut at 200 μm on a vibratome (VT1000S; Leica Microsystems), and small blocks of the dentate gyrus were trimmed from these sections. These specimens were cryoprotected, frozen at −190 °C in a cryofixation unit (EM CPC; Leica Microsystems), and then transferred to a cryosubstitution unit (EM AFS; Leica) which was programmed for all steps. Specimens were subsequently infiltrated with Lowicryl HM20 resin (Electron Microscopy Sciences), and the resin was polymerized as described previously.