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).
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