Quantitation of the labelling index for single-labelled MGMT (number of stained nuclei divided by total number of nuclei expressed as percentage) was carried out in the software program imageJ version 1.46e from the National Institute of Health (Rasband, W.S., http://rsbweb.nih.gov/ij/).
To examine the expansion of labelled cells more precisely, the number of labelled cells (clone size) within different compartments was determined at 3, 8 and 180 days of tracing.
At P120, mean signal intensity in RGCs was significantly lower in transgenic mice than in WT, whereas the CtB-labelled area (an indication of the number of labelled RGC) was similar (Fig 5A).
Furthermore, we intraperitoneally inoculated the BrdU-labelled P388 cells at 10 5), 10 6) and 10 7) cells per mouse into three groups of ten CDF1 mice and then quantified the distribution of the BrdU-labelled cells by counting the number of the labelled cells per unit area at each milky spot and non-milky spot site in the omentum.
Therefore, for any further analysis of labelled ICM cells, numbers were normalised against the total number of labelled ICM cells for each embryo (in percent) (see Materials and methods for details).
Labelling indices were obtained by counting cell number of labelled cells among at least 500 cells per region and expressed as a percentage values.
Labelling Index (LI) was quantified by determining the number of labelled cells/total number of cells counted × 100.
The distribution of the labelled cells along the crypts of the normal mucosa, was evaluated by recording the number of labelled cells in the three compartments of the crypt visible in longitudinally sectioned crypts, ideally divided into three equal parts: lower, mid and upper.
Estimates of the density of labelled cells were determined by examining coronal cortical sections at 400× and counting the number of labelled cells within comparable regions of the CC (corpus callosum) (four areas per hemisphere) and cortex (three areas, upper cortex and three areas, lower cortex per hemisphere).
