Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 paraformaldehyde at room temperature for 20 min, washed three times with PBS, and then permeabilised with 0.5% Triton X-100 in PBS at room temperature for 5 min. Neurons were then incubated in 50% normal goat serum in 1% BSA in PBS for 30 min at room tem- perature. After washing, neurons were incubated with primary antibody for 1 hour at room temperature, fol- lowed by a 45 min incubation with secondary antibody at room temperature. The following antibodies were used: mouse monoclonal phospho-c-Jun (ser63) anti- body (Santa Cruz), rabbit polyclonal activated caspase-3 antibody (Abcam), mouse monoclonal cytochrome c antibody (BD Biosciences, Oxford, UK), rabbit polyclo- nal MAP2 antibody (Millipore). Fluoroscein- or rhoda- mine-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Stratech, UK) were typically used at a dilution of 1:250. Neurons were rinsed in PBS and nuclei stained with DAPI dye in Antifade (DAKO) or Hoechst dye and mounted on glass slides. TUNEL stain- ing was performed using an in situ cell death detection kit (Roche) according to the manufacturer’s protocol. Microscopy and image collection Slides were viewed on a Zeiss Axioplan 2 microscope using a Plan-Apochromat 63x/1.40 oil objective. Images were captured at room temperature (20°C) using a Quantix digital camera (Photometrics, USA) and SmartCapture VP software. For the different treatments for each gene, the optimal exposure time was determined using the +NGF (0 hour) coverslip and was kept constant for all subsequent images for the remaining timepoints. For immunofluorescence time course experiments, all coverslips in each series for a particular gene were analysed in parallel and then saved as TIFF files and viewed using Adobe Photoshop CS4. Brightfield images were collected using a Zeiss Axiovert 200 M microscope with a Plan- Apochromat 63x/1.40 oil objective. The microscope stage was maintained at 37°C with 5% CO2. Images were captured using a Zeiss axiocam and Axiovision 4.0 software. Statistical analysis The statistical significance of differences between means was analysed by performing an unpaired Student’s T- test (for two-tailed distributions). To compare normal- ised data to a control sample, that has no error asso- ciated to it (for example, where +NGF is set to 1), the log10 values of the data were taken and a one sample T-test was used (for two-tailed distributions) as pre- viously described [57]. All data are presented as the mean ± S.E. of multiple experiments and significance is expressed as follows *P< 0.01. Page 15 of 17 Acknowledgements and Funding We would like to thank Nipurna Jina for sample proces- sing and quality control, Dr. Sonia Shah for help with the microarray analysis and Cephalon, Inc. for CEP- 11004. We would also like to thank Dr. Solange Desa- gher for critical reading of this manuscript. This work was supported by a Wellcome Trust Senior Research Fellowship awarded to JH (Grant number 057700) and a Wellcome Trust VIP award to MK. FM was funded by the Annabel McEnery Children’s Cancer Fund. We declare that there are no conflicts of interest. Additional material Additional file 1: All genes up-regulated by NGF withdrawal in this study. A PDF file listing the fold increase in RNA level after NGF withdrawal of all genes that are up-regulated more than 2-fold and have an FDR-adjusted p-value of < 0.01. Additional file 2: All genes down-regulated by NGF withdrawal in this study. A PDF file listing the fold decrease in RNA level after NGF withdrawal of all genes that are down-regulated more than 2-fold and have an FDR-adjusted p-value of < 0.01. List of abbreviations FDR: false discovery rate; GO: gene ontology; MLK: mixed lineage kinase; NGF: nerve growth factor; NS: not significant; PLIER: probe logarithmic intensity error estimation; ROS: reactive oxygen species; SCG: superior cervical ganglion; SE: standard error of the mean. Authors’ contributions RH carried out the real-time qPCR experiments. MK, FM and JH analysed and interpreted the microarray data. FM and MK performed the functional enrichment and gene ontology analysis and generated the heatmaps. MK performed all of the remaining experiments and analysis and wrote the manuscript. MH analysed the data and edited the manuscript. JH designed the study and wrote the manuscript. 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