PMC:3191169 (7) JSONTXT < >
|Materials and Methods|
|Image acquisition and statistics|
Pictures were acquired on a Zeiss Axiovert 200M or Zeiss LSM confocal microscope using an Axiocam camera and Axiovision software. We used 10x, 20x and 63x Zeiss objective lenses with numerical apertures of 0.3, 0.8 and 1.3 (oil), respectively. Pictures were further processed using Photoshop software (Adobe).
For data in Fig. 1 and Fig. S1, four independent experiments were performed. In each experiment 20 neurons/condition were analyzed. Filopodia number (>1 µm) and growth cone surface was determined for all growth cones/neuron using Axiovision software. For quantification of neurite number and branches, only neurites of more than 30 µm and branches of more than 2 µm were included. In Fig. 2, three independent cultures/condition were analyzed using Image Quant software (Molecular Dynamics). To evaluate ERK nuclear translocation (Fig. 3), four independent experiments including 25 neurons/experiment/condition were analyzed. A ratio of cytoplasmic vs. nuclear P-ERK intensity was determined using Axiovision software. In qRT-PCR, RNA of at least three independent cultures/condition was harvested.
Statistical significance was assessed by using a two-tailed t-test (Excel software) with *, **, *** representing P≤0.05, 0.01, 0.001. Error bars indicate standard deviation (s.d.) if not indicated otherwise.
|Unselected / annnotation||Selected / annnotation|