Taurine Effect on Stem Cells and Synaptogenesis Figure 9. The relative percentage of the effect of taurine on synaptic puncta and protein levels. Taurine was added to the primary neurons on day 2 and the neurons were then fixed and processed at day 9 and 15. The quantification of synapsin 1 and PSD 95 positive puncta was done by measuring the number of puncta per mm. Data are presented as percentages normalized to the control (100%) 6 SEM and puncta from at least 20 different neurons of each batch from at least three repeated experiments were quantified. Taurine treatment significantly increased the number of Synapsin 1 (A) and PSD 95 (B) puncta. Representative images show synapsin 1 (top panel) and PSD 95 puncta (bottom panel) respectively (C). The protein levels of synapsin 1 (D) and PSD95 (E) in protein lysates in primary neurons were shown using western blotting analysis (D-F). Data were normalized with total protein levels of a-tubulin. * P,0.05 and ** P,0.01. Scale bar = 1 mm. doi:10.1371/journal.pone.0042935.g009 objective using Zeiss LSM 710 confocal system (Carl Zeiss Pte Ltd, Singapore). The number of EdU-labeled cells that expressed DCX or GFAP was determined by counting a minimum of 35 EdU- labeled cells on the sections spanning the entire dentate gyrus as described previously. The extent of colocalization was validated by viewing cells on three planes (X, Y, and Z) using Z-plane sectioning. Cells single labeled for EdU or double labeled for EdU/DCX or EdU/GFAP were counted. The percentage of EdU cells double labeled for DCX or GFAP was calculated by dividing the number of double-labeled cells by the total number of EdU cells and multiplied by 100. Data are presented as percentages normalized to the control (100%) 6 SEM. Data were analyzed by t test. P,0.05 is considered as significant. For staining of embryonic brain, at E17, brains were fixed in 4% paraformaldehyde. Cryostat sections (20 mm) of the brain were cut, mounted onto gelatinized slides and processed for EdU chemistry. EdU staining is performed as described for the cultured hippocampal slices. To determine the EdU intensity in the dentate gyrus, 6 sections (250 mm apart) were selected from identical areas of hippocampus of vehicle or taurine injected embryos (n = 6). Images were taken with Zeiss LSM 710 confocal system (Carl Zeiss Pte Ltd, Singapore) using a 40X objective. For quantification, a square of 250 mm2 was randomly placed on three different locations of the DG and images were taken maintaining the same imaging settings for all conditions. The fluorescence was quantified using image J. For each slice, three squares were analyzed and the three values were averaged/slice. Data are presented as percent- ages normalized to the control (100%) 6 SEM and statistical analysis was done using t test. P,0.05 is considered as significant. Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot Analysis Protein concentrations were determined using the BioRad Protein Assay. Dye Reagent Concentrate (Bio-Rad; Hercules, CA, USA). Equal amounts of samples were separated by SDS-PAGE. Following electrophoresis, the gel was transferred to a polyvinyldi- fluoride (PVDF) membrane (Millipore, Bedford, MA, USA), blocked with 5% skim milk in PBST (0.1 M PBS and 0.1% Tween 20), then incubated with the desired antibody at 4uC overnight (1:5000 for the anti-p44/42 antibody (Cell Signaling), 1:1000 for the anti-Phospho-P44/42 antibody (Cell Signaling), 1:1000 for synapsin 1 (Abcam), 1:300 for PSD 95 (Abcam), and 1:5000 for the anti-a-tubulin antibody (Sigma). After three 10-min washes in PBST, the membranes were incubated with peroxidase- conjugated donkey anti-rabbit, goat anti-mouse IgG (Promega), at a 1:5000 dilution for 1 h at room temperature (RT) and washed six times with PBST. The immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) method. The data were analyzed using image-J software. One-way ANOVA with Dunnet’s post hoc test was used to analyse data on ERK1/2 and a t test was used to analyze western blot on synaptic proteins. P,0.05 is considered as significant. Acknowledgments We thank Associate Professor Tang Bor Luen for critically reading the manuscript and essential advice, Dr Samuel Ko and Dr Daniel Koch from Carl Zeiss, Singapore for imaging support on Zeiss LSM 7 ELYRA PS.1 system, Eddie Tan and Wan Ying Leong for technical support, members of the Goh lab for sharing of reagents and expertise. PLOS ONE | www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e42935