Rolera mgi camera

VD growth cones were imaged as previously described (Norris et al., 2009 (link)). Briefly, animals harboring the indicated transgenes were selected 16 h post-hatching at 20°C and placed on a 2% agarose pad with a drop of 10 mM muscimol (Sigma-Aldrich) in M9 (Weinkove et al., 2008 (link)), which was allowed to evaporate for 4 min before placing a coverslip over the sample. Growth cones were imaged with a Qimaging Rolera mGi camera on a Leica DMR microscope. Images were acquired at intervals of 120 s, with total duration of time-lapse ranging from 20 to 60 min.
Dynamic projections less than 0.5 µm in width emanating from the growth cone were scored as filopodia. Maximal filopodia length was measured using ImageJ software, and filopodial duration was determined by persistence of the protrusion through time-lapse images. All filopodia on multiple growth cones were analyzed, and at least seven growth cones of each genotype were included in the analysis (at least 25 filopodia). In Fig. 2E, the average length of filopodia was determined from images of growth cones (at least ten growth cones; at least 25 filopodia). The significance of differences was determined by a two-sided t-test with unequal variance.

VD growth cones were imaged and quantified as previously described [22 (link)]. Briefly, animals at ~16 h post-hatching at 20°C were placed on a 2% agarose pad and paralyzed with 5mM sodium azide in M9 buffer, which was allowed to evaporate for 4 min before placing a coverslip over the sample. Some genotypes were slower to develop than others, so the 16 h time point was adjusted for each genotype. Growth cones were imaged with a Qimaging Rolera mGi camera on a Leica DM5500 microscope. Images were analyzed in ImageJ, and statistical analyses done with Graphpad Prism software. As described in [22 (link), 23 (link)], growth cone area was determined by tracing the perimeter of the growth cone body, not including filopodia. Average filopodial length was determined using a line tool to trace the length of the filopodium. Unless otherwise indicated, ≥25 growth cones were analyzed for each genotype. These data were gathered in ImageJ and entered into Graphpad Prism for analysis. Analysis of Variance (ANOVA) was used to determine significance of difference between genotypes. Any of the VD growth cones visible at the time of imaging were scored (VD2-VD13), and we did not focus on any single VD growth cone for analysis.

VD growth cones were imaged as previously described [15 (link), 22 (link)]. Briefly, animals at 16 h post-hatching at 20°C were placed on a 2% agarose pad and paralyzed with 5mM sodium azide in M9 buffer, which was allowed to evaporate for 4 min before placing a coverslip over the sample. Some genotypes were slower to develop than others, so the 16 h time point was adjusted for each genotype. Growth cones were imaged with a Qimaging Rolera mGi camera on a Leica DM5500 microscope. Projections less than 0.5 µm in width emanating from the growth cone were scored as filopodia. Filopodia length and growth cone area were measured using ImageJ software. Filopodia length was determined by drawing a line from the base where the filopodium originates on the edge of the peripheral membrane to the tip of the filopodium. Growth cone area was determined by tracing the periphery of the growth cone, not including filopodial projections. Significance of difference was determined a two-sided t-test with unequal variance.