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. 2016 Jan 26;21(12):1752–1767. doi: 10.1038/mp.2015.212

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Copyright © 2016 Macmillan Publishers Limited

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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Effects of erythropoietin (EPO) on neuronal differentiation and maturation, determined by mean active area of synapses in E17-HCC. Neurons (d8) were quadruply stained for differentiation markers Dcx and MAP2 and activity markers Synaptotagmin I (SytI) and Synapsin I (SynI) (n=8). Staining was analyzed by confocal microscopy. (a) The protocol includes three sequential scans with fixed emission windows (orange) with different excitation wave length. Excitation (dashed line) and emission (solid line) spectra are shown for each fluorescent dye, Alexa Fluor 488 (MAP2, green), Alexa Fluor 546 (SynI, blue), Alexa Fluor 594 (SytI, red) and Alexa Fluor 633 (Dcx, black). 4,6-Diamidino-2-phenylindole (DAPI) fluorescence was determined using an additional excitation at 406 nm. (b) Unmixed confocal picture showing differentiation markers Dcx (red), MAP2 (green) and DAPI (blue). (c) Quantification of the integrated density of Dcx and MAP2 presented as ratio (n=8 per group, paired one-tailed t-test). (d) Unmixed confocal picture showing SytI (green) and SynI (red). (e) Higher magnification of the square mark of picture d, showing single stained dots for SytI (green, arrowhead), single stained dots for SynI (red, star) and colocalized dots (yellow, arrow). (f) Masked images for analyzing the number and area of SytI (green, arrowhead), SynI (red, star) and colocalized (white, arrow) dots. (g) Quantification of the mean overlapping (colocalized) area as mean active area (white spots in f, n=8 per group, paired two-tailed t-test). All n-numbers given are derived from biological replicates, that is, independent cell preparations. All bar graphs are shown as mean±s.e.m.; *P<0.05.