Fig. 3

Media from microglia overexpressing Sema 3A cause axonal retraction and increase dendritic arborization in NP within an hour of exposure. A Representative Ank G (green) and β-III tubulin (red) staining analysis of NP after 60 min and 180 min exposure to media from microglia overexpressing Sema 3A (Sema 3A), or GFP empty vector (GFP) or non-transfected microglia (Ctrl). Scale bar: 20 µm. Figures were acquired by 40 × objective and cropped in order to visualize each single neuron. Skeleton mask is reported below. Scale bar 10 µm.The corresponding axonal length measure is reported in B. Analyses were performed using Neuron J plug-in. Data are the mean ± SEM of four independent experiments performed in duplicate (approximately 10 neurons for each slide). One-way ANOVA followed by Tukey’s test for multiple comparisons. *P < 0.05; ****P < 0.0001. Representative MAP-2 staining is shown in C. Scale bar 50 µm. D Dendritic branch measurements (Sholl analysis) of NP exposed to media from Ctrl, Sema 3A and GFP microglia. Data are the mean ± SEM of three independent experiments performed in duplicate (approximately 10 neurons for each slide). Analyses were performed using Neuroanatomy plug-in of ImageJ software. Two-way ANOVA followed by Tukey’s test for multiple comparisons. *P < 0.05 vs Ctrl. E Dendrite clustering analysis according to Strahler's order. Analyses were performed using Neuroanatomy plug-in of Image J software. Data are the mean ± SEM of three independent experiments performed in duplicate (approximately 10 neurons for each slide). Two-way ANOVA followed by Tukey’s test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001: ****P < 0.0001