solution for 40 min at 37uC. After washing in TBST (0.14 M NaCl, 10 mM KCl, 25 mM Tris, pH 7.0, and 0.1% Tween-20), endogenous AP was inactivated by incubating the spinal cords in TBST for 20 min at 65uC. The spinal cords were then incubated in blocking solution (10% goat serum, 1% BSA in PBS) followed by an overnight incubation at 30uC with an anti-DIG-AP conjugate (Roche Diagnostics, Indianapolis, IN, USA; diluted 1:2000 in blocking solution). They were then extensively washed in MABT, stained as described for the whole-mount in situ hybridization [27], and examined under transillumination. Several spinal cords from wildtype and mutant embryos, issued from the same litter, were examined. TUNEL positive cells were counted in the lumbar region specifically as previously described [30]. Spinal cord explants Lumbar spinal cord regions were dissected from E12.5 (somites counted) wildtype or Necdin-deficient embryos issued from the same litter. We excluded the ventral part of the dorsal root ganglia with the corresponding part of the sclerotome. From each lumbar region dissected, two sections were made to create three explants. The dissection and culture of explants was performed as previously described [34]. Briefly, these explants were grown in four-well culture dishes on coverslips previously coated with polylysin/ laminin in Neurobasal medium containing 20% FBS and NGF (100 g/ml). Culture medium [34] was complemented with (+NTF) or without (2NTF) a cocktail of NTFs (GDNF, 1 ng/ml; CNTF, 10 ng/ml and BDNF, 10 ng/ml). After two days in vitro (DIV), explants were fixed for 10 min at RT in 4% paraformaldehyde in PBS, washed 3 times in cold PBS, and incubated overnight in 20% sucrose at +4uC. The explants were then frozen in a Tissue-Teck cryostat (Oxford laboratories) and stored at 280uC. Cryosections (12 mm) were performed for each animal (3 explants/lumbar spinal cord) and immunolabeled with anti-Islet-1/-2 antibodies as indicated above. An average of 25 sections was obtained for each explant. The number of Islet-1/-2 positive cells was counted for each section and the mean of these values (25 sections63 explants) was calculated for each lumbar spinal cord. Values in Figure 4B represent the mean values of sections per animal. Cell survival For motoneuron quantification in vivo, spinal cord sections (10 mm each, collected every fourth sections) were immunostained with the motoneuron marker Islet-1/-2 as indicated above. Only the cells located in the ventral part were considered as motoneurons. The interneurons located in the medio-dorsal part were separately counted at E17.5. For caspase-3 quantification in the lumbar region in vivo, the number of cleaved caspase-3 positive cells was counted on each transversal section (10 mm), and the mean number of caspase-3 positive cells was determined per section. For the quantification of cleaved caspase-3, counting was done directly on whole mount spinal cords immunolabeled with anti-cleaved-caspase-3 antibod- ies. For motoneuron culture, E12.5 Necdin+/+ and Necdin+/2 embryos were collected from the same mother and genotyped by PCR as described above prior to dissection of the spinal cord. For Hb9::GFP motoneuron cultures, E12.5 transgenic embryos were sorted under a fluorescence microscope prior to dissection of the spinal cord. Embryonic motoneurons were isolated as described [54,55], using iodixanol density gradient centrifugation. Motoneurons were plated on poly-ornithine/laminin-treated wells in supplemented Neurobasal medium at a density of 1,100 cells/ cm2 for survival assays and 2,600 cells/cm2 for immunofluorescent labeling. Neurons were cultured in the presence (or not, when Necdin Protects Motoneurons from Death indicated) of a cocktail of NTFs (GDNF, 0.1 ng/ml; BDNF, 1 ng/ ml and CNTF 10 ng/ml) [54,55]. When indicated motoneurons were treated at indicated times with 100 ng/ml of TNFR1-Fc (Alexis Biochemicals, San Diego, CA, USA) or 100 ng/ml of TNFa (BD Biosciences, Franklin Lakes, NJ, USA) and survival was determined at indicated times under light microscopy using morphological criteria [54]. To compare values from different experiments in a quantitative manner, the number of motoneu- rons cultured in the presence of neurotrophic factors was taken as 100% survival. Neurite outgrowth analysis Neurons were fixed three days post seeding with 4% paraformaldehyde in PBS and immunostained with rabbit polyclonal anti-Tau antibodies (ab64193, Abcam, Cambridge, MA, USA, 1:200) labeling being revealed using a Cyanine 3 anti- rabbit antibody (Jackson Immunoresearch, 1:500) and a mouse monoclonal anti-GFP (NB600-597, Interchim, 1:500) antibody revealed using an Alexa anti-mouse antibody (1/1000). Based on the immuno labeling and using Metamorph software, the total length of neurites was measured and compared between mutant and wildtype motoneurons. Western blotting Lumbar ventral spinal cords were dissected at different embryonic and postnatal developmental stages from wildtype and Necdin mutant mice. Tissues were snap frozen in liquid nitrogen and stored at 280uC. Protein extractions were performed as indicated previously [56]. Homogenate samples containing equal amount of proteins (30 mg as determined using BCA Protein Assay Reagent, Thermo scientific, Waltham, MA,USA) were boiled for 5 min in SDS buffer (50 mM Tris-HCl, pH 6.6, 2% SDS, 10% glycerol, 0.1% bromophenolblue, and 5% b-mercap- toethanol), and separated on a 10–20% SDS/polyacrylamide gel (Biorad) in Tris-Tricine buffer. After migration, proteins were transferred onto 0.2 mm nitrocellulose membranes (Millipore). Membranes were blocked for 1 hour at RT with TBS-T buffer (150 mM NaCl, 20 mM Tris HCl, pH 7.4, 0.1% Tween-20) containing 5% milk. The membranes were then incubated overnight with polyclonal antibodies against Necdin diluted at 1:500 in TBS-T containing 2.5% milk. Membranes were washed three times in TBS-T and incubated for 1 hour with an anti-rabbit horseradish peroxydase antibody. The proteins were visualized by enhanced chemiluminescence (ECL plus, GE Healthcare, Buck- inghamshire, UK). Each blot was re-probed using an anti mouse anti-Actin (Sigma-Aldrich, St Louis, MO, USA) antibody, as loading control. Statistical analyses According to the sample size, we used non-parametric statistical analysis. Statistical comparisons between independent samples were made using the Wilcoxon-Mann-Whitney rank sum test (StatXact software, Cytel Inc., Cambridge, MA, USA). Values are indicated as following: (Q2 (Q1, Q3), n, P value), where Q2 is the median, Q1 is the first quartile and Q3 is the second quartile. The level of significance was set at a P value less than 0.05. Supporting Information Figure S1 The absence of Necdin leads to motor deficit in mice. (A) At P8, a difference in the body size and in position of the hindpaws is observed between wildtype (Necdin+/+) and Necdin- deficient (Necdin+/2) pups. The Necdin+/2 pups are unable to flex and move their hindlimbs. (B) In adulthood, the Necdin-deficient PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e23764