mNgn2-induced neurons and control GFP transduced NPCs at two or three weeks of culture were immunostained with MAP2AB and DAPI and mounted, as described in Sections 2.3.1 and 2.3.2. MAP2AB and DAPI images were captured (five images each from three biological replicates per condition and cell line) using an epifluorescence microscope (Olympus IX51) at 100× total magnification. DAPI images were thresholded, and cell segmentation was achieved using the watershed separation feature in NIH Image J (http://imagej.nih.gov/ij/), so that DAPI-positive nuclei could be counted using NIH Image J. The MAP2AB area was measured using NIH Image J, and then divided by DAPI-positive nuclei numbers in order to calculate MAP2AB levels normalized to cell count.
