Eclipse te600 microscope

To evaluate neuritic outgrowth and connectivity, fixed cells were stained with the following primary antibodies: a rabbit polyclonal anti-MAP2 (1:100, Merck Millipore, Burlington, MA, USA) and a mouse monoclonal anti-PSD-95 (1:100, Abcam, Cambridge, UK) antibody. Detection was performed using a FITC-conjugated anti-rabbit IgG (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and a Cy3-conjugated anti-mouse IgG (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) antibody, respectively. Nuclei were counterstained with Hoechst-33342 (Sigma-Aldrich, St. Louis, MO, USA) and fluorescent images were acquired using a Nikon Eclipse Te600 microscope equipped with a Nikon Digital Camera DXM1200 ATI system (Nikon Instruments, Inc., Melville, NY, USA). The neuritic length of MAP2-positive neurons was measured and quantified by tracing along each neuronal projection using the image analysis system Image Pro Plus (Media Cybernetics, Silver Spring, MD, USA) as previously described [11 (link)]. The degree of synaptic innervation was evaluated by counting the number of PSD-95 positive puncta on proximal dendrites and expressed as the number of PSD-95 puncta per 20 μm of neuritic length. Immunofluorescence images were taken with a LEICA TCS SL confocal microscope (Leica Mycrosystems, Wetzlar, Germany). Fifty neurons for each condition were evaluated.