Axon length and growth cone analysis

For axon length measurements, the length of 100 neurons per transfection per experiment (n = 3) were measured using neuron J, selecting healthy neurons with an uninterrupted axon that was at least 3 times the length of the cell body.

The growth cones of neurons labelled with anti-GFP and anti-tubulin antibodies were imaged using a Zeiss AxioScop at 63X, and images were processed using Volocity or ImageJ software. ImageJ was used to quantitate growth cone area, by thresholding the GFP fluorescence, drawing an outline and measuring the area in pixels. A total of ~70 neurons per construct were analysed from 3 independent experiments. The graphs were drawn based on the average area and Standard error of mean.