4.13. Quantification of cell number and positions

The number of phospho-histone H3⁺ cells per section was quantified manually and obtained data were plotted and analysed statistically using GraphPad Prism v. 5.

The number and position of PCNA⁺ (or Hu⁺), foxj1a:GFP⁺ and DAPI⁺ cells in the injured spinal cords were quantified using the 3D Objects Counter plugin available in Fiji [54]. Briefly, a maximum projection image was generated from three optical sections with 1 µm between sections. The image of the DAPI channel was then used to automatically segment individual nuclei using the plugin. In nuclei that overlapped and were not segmented automatically, the boundary between nuclei was defined manually. The segmentation mask that identified individual objects (nuclei) in the DAPI channel was then used to extract the fluorescence intensity in the other two channels (GFP and PCNA/Hu). The plugin output included the position of each nucleus and the average fluorescence intensity in the GFP and PCNA/Hu channels. We defined that a nucleus was positive for a specific channel if the average intensity was above the background levels (measured manually). Matlab was used to analyse the obtained data and to generate scatter plots of cell positions for each time-point after injury (composite maps).

GraphPad Prism 5 was used to plot the total number of different cell types per section, to compare between different conditions and different time-points post-injury and to perform statistical analysis.