Taurine Effect on Stem Cells and Synaptogenesis Figure 8. The effects of taurine on neurite outgrowth of NPCs and primary neurons. The average total neurite length (A and D) and neurite numbers (B and E) were measured. Images in C and F show representative primary neurons and NPCs treated with vehicle (left panel) or taurine (right panel) respectively. Taurine was added to the media on day 2. Primary neurons and NPCs were then fixed on day 5 and 7 respectively. Differentiated neurons were then analyzed and data presented as mean 6 SEM. Taurine shows a small non-significant effect on neurite development of primary neurons as well as newborn neurons. Scale bar = 20 mm. doi:10.1371/journal.pone.0042935.g008 throughout every medium change. EdU (10 mM) was added to the culture medium on day 11 for 48 hrs and the slices were fixed on day 17 using 4% phosphate buffered paraformaldehyde and then processed for EdU, DCX and GFAP staining. Data were analyzed by one-way ANOVA with Dunnet’s post hoc test. P,0.05 is considered as significant. Animals Time-mated C57BL/6 mice and Sprague-Dawley rats were purchased from Singhealth Experimental Medicine Centre (SEMC), Singapore, and housed in Specific Pathogen Free (SPF) animal facility at Duke-NUS Graduate Medical School, Singa- pore. All animals received water and food ad libitum. All animal procedures and applicable regulations of animal welfare were in accordance with IACUC guidelines. For labeling of proliferating cells in utero, a single injection of EdU (Invitrogen) dissolved in PBS were given at a dose of 50 mg/kg body weight on E13, 1 hr before the intra uterine taurine or vehicle injection. Immunocytochemistry, Immunohistochemistry, Confocal Imaging and Analysis The following primary antibodies were used: goat anti-DCX (Santa Cruz, 1:500), rabbit anti GFAP (Abcam, 1:100). Rabbit anti-synapsin 1 (Abcam, 1:500), mouse anti-PSD95 (Abcam, 1:300). Images were acquired on a Zeiss LSM 7 ELYRA PS.1 system (Carl Zeiss, Pte. Ltd., Singapore) and analyzed using Zeiss Zen software. For analysis of the dendritic structure of neurons, the images were semi-automatically traced with NIH ImageJ using the NeuronJ plugin. The total dendritic length and branch number of each individual neuron were analyzed. A total of 200 neurons were analyzed per group. The synapse puncta was calculated as the number per mm. Data are presented as percentages normalized to the control (100%) 6 SEM and at least 15 different neurons were quantified for each group and at least three repeated individual experiments were done. For immunohistochemical analysis of brain slice cultures, slices were cryoprotected in 20% and the 30% sucrose solutions over 2 days. Next, the slices were embedded in an OTC compound and sectioned on a cryostat at thickness of 30 mm. After permeabilizing and blocking with serum, the sections were incubated with EdU staining reaction mix (Invitrogen) for 1 hr. Then the sections were incubated with antibodies against DCX and GFAP overnight at 4uC. The sections were then washed and incubated with secondary antibodies. To determine the number of EdU positive cells in the granule cell layer (GCL) including the subgranular zone and the hilus of cultured slices, stacks of optical sections were taken under a confocal laser-scanning microscope using a 40X objective. For quantification, a square of 250 mm2 was randomly placed on three different locations of the DG. The cells in each square were counted and the three counts were averaged/section. Adjacent sections were not used for cell counting to avoid double counting. A total of 5 sections per cultured slice were used, and each experimental group consisted of 15–17 cultured slices from 3 independent experiments. Double-labeled cells for EdU and DCX or GFAP were visualized and counted with a 63 X oil immersion PLOS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e42935