Amoureux et al. BMC Cancer 2010, 10:91 http://www.biomedcentral.com/1471-2407/10/91 For studying the effect of PSA-NCAM on differentia- tion, cells were treated with endoN (1/1000) for 3 days in vitro. GBM9 cells were fixed with 4% paraformalde- hyde for 15 minutes at room temperature (RT), and washed with PBS. Cells were permeabilized with 0.1% Triton X-100 and 3% BSA for 45 minutes. Primary anti- bodies were incubated for 2 h at RT in PBS. Following 3 washes of 10 minutes each, appropriate secondary anti- bodies were incubated for 1 h at RT. Nuclei were stained with DAPI (0.5 μg/ml) for 5 minutes and washed with PBS. Immunofluorescence staining was observed using the Carl Zeiss Observer D1 inverted microscope (Carl Zeiss, Gottigen, Germany) equipped with a 20 × objective. The following antibodies were used: olig2 (rabbit IgG, 1/1000; Abcys), GFAP (mouse IgG, 1/1000; Millipore, Molsheim, France), b3-tubulin (mouse IgG, 1/1000; Sigma), Nestin (mouse IgG, 1/500; Abcys), PSA-NCAM (mouse IgM, 1/1000; Abcys), Alexa555-anti-mouse IgG, CY3-anti-mouse IgM, Alexa488-anti-rabbit IgG (all 1/500; Jackson Laboratory). Western Blot C6 and C6-PSA-NCAM total cell extracts were prepared by scraping cells in 500 μl of 10 mM TrisHCl pH 7.4 containing 0.1% TritonX-100 and Complete™ protease inhibitors (Roche, Mannheim, Germany) followed by sonication. Proteins were run on an 8% SDS-polyacryla- mide gel. Western blot was performed according to standard protocols with anti-olig2 (1/4000), anti-PSA- NCAM (1/1000), and anti-a-tubulin (mouse IgG, 1/ 2000, Sigma). Secondary antibodies were coupled to HRP (1/20000; Jackson Laboratory). Statistical analysis The percentage of olig2 positive cells in GBM9 differen- tiation assay was analyzed using contingency tables fol- lowed by the Fisher’s exact test. The Spearman correlation coefficient was used to evaluate the consis- tency between the 2 methods of PSA-NCAM content measurements in biopsies (immunohistochemistry and ELISA). For statistics performed on GBM patients, the SPSS 15.0 software for windows (SPSS Inc, Chicago, Illinois, USA) was used. Survival was estimated using the Kaplan-Meier method, and curves were compared using the log-rank test. On multivariate analysis, the effect of potential risk factors including age (< 60 versus ≥ 60), preoperative KPS (< 70 versus ≥ 70), extent of surgical excision (partial versus total), and PSA measurement by ELISA (≤ 10 pg PSA-NCAM/μg of protein versus > 10 pg PSA-NCAM/μg of protein) on the DFS and OS were evaluated using the cox regression proportional hazard models. The threshold for statistical significance was p = 0.05. Page 4 of 12 Results PSA-NCAM ELISA sensitivity, linearity and specificity We developed a sandwich ELISA to detect PSA-NCAM by taking advantage of the monoclonal anti-PSA-NCAM developed in our laboratory [21] (Abcys) as the capture antibody. An anti-mouse NCAM was then produced by immunizing goats with NCAM purified from adult mice brain, and used as the detection antibody. A linear relationship could be established between known amounts of PSA-NCAM and optical density readings between 0.25 and 16 ng PSA-NCAM/ml (Fig- ure 1A). Tissue extracts were tested after dilution at 37 μg of protein/ml; therefore the corresponding limit of detection in such sample is 6.75 pg PSA-NCAM/μg of protein. The test was linear over 2 logs around the con- centration tested. Intra-assay coefficient of variation was less than 5% (n = 3). Although the ELISA was designed with appropriate antibodies to detect PSA-NCAM, we Figure 1 Standard curve of PSA-NCAM ELISA. (A) Increasing amounts of purified PSA-NCAM from P0 mouse brains were detected by the developed ELISA. Data are presented as mean +/- standard deviation of duplicate measurements. (B) ELISA test on serial dilutions of TE671 extract expressing PSA-NCAM or treated with endoN.