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. 2019 Nov 1;13:486. doi: 10.3389/fncel.2019.00486

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Copyright © 2019 Colnaghi, Russo, Natale, Restelli, Cagnotto, Salmona, Chiesa and Fioriti.

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(s) 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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Confocal microscopy and SIM analyses of DIV18 primary hippocampal neurons to determine the localization of SUMO2/3, synaptophysin and PSD95. (A) Confocal microscopy of primary neurons. Cells were immunostained for SUMO2/3 using our custom antibody (green), synaptophysin (red) and Map2 (magenta). DAPI was used to stain the nuclei. Scale bar, 50 μm. Images were obtained using a 40× objective and displayed as z projection. (B) SIM analysis using a 100× objective. Colored arrowheads indicate the position of the inset shown in panel (C). (C) The Merge images represent single stack of SUMO2/3 (green) and synaptophysin (red). Scale bar, 0.5 μm. (D) Intensity profile normalized for each channel to 100 (arbitrary unit) using the same color code of SIM merge images and representing the values indicated by the cyan arrow. (E) Confocal microscopy of primary neurons. Cells were immunostained for SUMO2/3 (custom antibody, green), PSD95 (red) and Map2 (magenta). DAPI was used to stain the cell nuclei. Scale bar, 50 μm. Images were obtained using a 40× objective and displayed as z projection. (F) SIM analysis using a 100× objective with colored arrows that indicate the position of the inset shown in panel (G). (G) Merge channel represent single stack image of SUMO2/3 (green) and PSD95 (red). Scale bar, 0.5 μm. (H) Intensity profile normalized for each channel to 100 (arbitrary unit) using the same color code of SIM merge images and representing the values indicated by the cyan arrow. (I) Pearson Correlation Coefficient between SUMO2/3 (custom antibody) and PSD95 (blue) and synaptophysin (SYN) (red). (J) SUMO2/3 fraction that colocalizes with PSD95 or synaptophysin (SYN) (M1) and PSD95 or synaptophysin fraction that colocalizes with SUMO2/3 (M2). Date are the mean ± SD of 40 fields from four independent experiments.