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. 2018 Jun 26;12:389. doi: 10.3389/fnins.2018.00389

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Copyright © 2018 Verstraelen, Van Dyck, Verschuuren, Kashikar, Nuydens, Timmermans and De Vos.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Validation experiments for synapse screening. (A) Validation of antibodies via western blot. A Synaptophysin (Syph) antibody shows the expected single band at 38 kDa. Conversely, a PSD-95 antibody shows, next to a bright band at the expected height, additional bands, indicative of non-specific binding (cort/hipp: 14 DIV mouse cortical or hippocampal primary neurons). (B) Validation of the spot detection algorithm using simulated images with increasing background (BG) and decreasing signal-to-noise ratio (SNR). Besides background and SNR levels, spot size and density determine the lower detection limit. (C) Spot detection algorithms can also be validated by comparison with a ground truth, obtained by manual spot counting in (regions of) real images. The binary images show the detection of spots after manual (M) or automatic (A) detection. Manual spot quantifications may differ (here lower) with those from the spot counting algorithm, but if counts change proportionally in low- and high-density regions, relative comparisons are still possible. Scale bar: 5 μm. (D) Validation of colocalization analysis for mature synapses by different marker combinations. The lower detection limit can be pinpointed using an inhibitory presynaptic (e.g., vGAT) and excitatory postsynaptic (e.g., AMPA-R) marker, which only colocalize by coincidence. The upper limit can be determined using two different primary antibodies for the same marker (e.g., synaptophysin). The combination of a pan-presynaptic marker (e.g., synaptophysin) and a specific postsynaptic marker (e.g., AMPA-R) should in turn yield an intermediate level of colocalizing spots, depending on the ratio of excitatory/inhibitory synapses. Scale bar: 5 μm. (E) Analysis of cultures at different days in vitro (DIV) can be used as a positive control for increasing connectivity, exemplified by an increasing density of pre- and postsynaptic spots. The number of mature synapses also increases, but is markedly lower compared to the pre- and postsynaptic markers alone. Scale bar: 10 μm.