Nanogold

All steps were carried out at room temperature unless otherwise indicated. Samples were made permeable and blocked with 0.1% saponin and 5% normal goat serum in PBS for 40–60 min, incubated with primary and then secondary antibodies (Nanogold, at 1:200, Nanoprobes, Yaphand, NY) for 1 hr, fixed with 2% glutaraldehyde in PBS for 30 min, and stored at 4˚C in fixative for up to 2 wks. Samples were washed thoroughly in deionized water, silver enhanced (HQ kit, Nanoprobes), treated with 0.2% osmium tetroxide in 0.1M phosphate buffer at pH 7.4 for 30 min, en block stained with 0.25% uranyl acetate in acetate buffer at pH 5.0 for 1 hr at 4˚C, dehydrated in graded ethanols, and embedded in epoxy resin.

Ultrastructural localization of GFP-tagged proteins in fixed transfected KB cells was performed by using anti-GFP immuno-TEM, combining nanogold (Nanoprobes, USA) labeling with silver enhancement (HQ Silver Kit, Nanoprobes, USA) [57 ]. To detect GFP-positive sites in TEM we used the following antibodies: (i) anti-human GFP mouse monoclonal (Roche Diagnostics); (ii) goat anti-mouse (Jackson, USA); (iii) streptavidin-nanogold conjugate (Nanoprobes). Cells were harvested without osmication by meticulous scraping from the glass surface, then dehydrated and embedded in epoxy resin as described in the previous section. Ultrathin sections were picked up on 200 mesh copper grids (EMS) and stained with uranyl acetate and lead citrate and examined and imaged as described above (for details see S4 Method).

EM was performed using standard procedures as previously described (Trimbuch et al., 2009 (link)). Briefly, following animal perfusion with 4% PFA, 0.1% glutaraldehyde, and 15% picric acid in 0.1 M phosphate buffer (pH 7.4), brains were isolated, postfixed, and cut on a vibratome in 50-µm thick slices. Following a freeze–thaw protocol, sections were incubated with rabbit-anti-IL-4Rα (Abcam) for 96 h at 4°C and subsequently with goat-anti-rabbit secondary antibody conjugated to 1.4 nm Nanogold (Nanoprobes) for 96 h at 4°C, followed by embedding in EPON 812 resin. Ultrathin slices (60 nm) of the stratum radiatum of the hippocampal CA1-region were cut on a Leica UCT ultramicrotome and imaged on a transmission electron microscope Zeiss 912. For assessment of presynaptic vesicles, animals were perfused with 2% PFA, 2.5% glutaraldehyde, and 15% picric acid in 0.1 M phosphate buffer (pH 7.4) and processed for fine structural analysis as described above. Quantitative analysis of presynaptic vesicle distribution was performed in 50 nm bins from the synaptic cleft. A total of 72–73 synapses were analyzed for each genotype (n = 6 cre⁺ animals and n = 6 cre⁻ animals).

Mouse anti-human IgG₁ CD63 (clone H5C6, catalog number 556019, 5 μg/mL, BD-Pharmingen, San Diego, CA), mouse anti-human CD9 (clone 209306; R&D Systems, 10 μg/mL, Minneapolis, MN) and irrelevant isotype control monoclonal antibodies (mAbs) were used for electron microscopy immunodetection studies. The secondary Ab for immunoEM was an affinity-purified goat anti-mouse Fab fragment conjugated to 1.4 nm gold particles (1:100, Nanogold, Nanoprobes, Stony Brook, NY). FITC-conjugated mouse anti-human IgG₁ CD63 (clone H5C6, Biolegend, San Diego, CA), FITC-conjugated mouse anti-human IgG₁ CD9 (clone HI9a, Biolegend), and irrelevant FITC-conjugated isotype control antibodies were used for nanoscale flow cytometry or regular flow cytometry.

Fixed samples were washed, and most were then blocked and made permeable with 5% normal goat serum and 0.1% saponin in PBS for 30-60 min. Some samples for labeling with the NR1, NR2B, and GluR2 antibodies were permeabilized with 50% ethanol for 10 min, and then were treated with 5% normal goat serum in PBS for 20-30 min. All steps were performed at room temperature unless otherwise indicated. Samples were incubated with primary and secondary antibodies (Nanogold, Nanoprobes) for 1 h, fixed with 2% glutaraldehyde in PBS for 30 min, then held at 4˚C, washed in water, and silver enhanced (HQ Kit, Nanoprobes), treated with 0.2% osmium tetroxide in 0.1 m phosphate buffer at pH 7.4 for 30 min on ice, en bloc stained with 0.25-0.5% uranyl acetate in acetate buffer at pH 5.0 for 1 h at 4˚C, dehydrated in graded ethanols, and embedded in epoxy resin. Controls for immunolabeling include omitting the primary antibody or comparison between different primary antibodies for specific labeling for different structural entities.

After treatment, neuronal cultures were processed for pre-embedding immunogold-labeling as described previously [24] (link). Briefly, cultures were fixed in 4% paraformaldehyde (EMS, Hatfield, PA) in PBS for 30–45 min at room temperature, permeabilized and blocked in 0.1% saponin and 5% normal goat serum for 40–60 min respectively, incubated with primary and secondary antibodies (Nanogold, Nanoprobes, Yaphank, NY) for 1–1.5 h, then fixed with 2% glutaraldehyde in PBS, silver enhanced (HQ kit, Nanoprobes), and processed for electron microscopy [24] (link). Only parallel samples from the same experiment were directly compared because the overall labeling sensitivity may differ between experiments.