Animals were rinsed again in PBS (2 × www.selleckchem.com/Wnt.html 2 min) and then fixed for 20 min in 4% paraformaldehyde. Animals were then rinsed in PBS-TX and labeled successively with goat anti-HRP overnight and donkey anti-goat for 1 hr at room temperature. For anti-HRP labeling without detergent, goat anti-HRP at 1:200 in PBS was added for 1 hr at room temperature, the animals were rinsed for 30 min in PBS, 30 min in PBS-TX, then incubated with other primary antibodies and secondary antibodies in PBS-TX each overnight at 4°C. Arbors were traced in Neurolucida (Microbrightfield, Natick, MA) and analyzed in Neurolucida Explorer. For time-lapse analysis, neurons were

quantified using the Simple Neurite Tracer plugin for FIJI. Arbors were traced as stacks (class I) or confocal projections (class IV). Line

scan analysis was performed using Metamorph software (Molecular Devices, Downingtown, PA). For determination of HRP immunoreactivity in relation to Coracle labeling in class IV neurons, regions of arbors were categorized as either “high Coracle” (n = 24 regions) or “low Coracle” (n = 15 regions) in confocal projections. Cumulative average fluorescence intensities were 137 arbitrary units (A.U.) for high Coracle regions and 66 for low Coracle regions. Regions for line scan analysis were selected as informative if no Venetoclax cell line Coracle-rich epidermal cell membranes intersected the line scan and no other dendrites crossed the line scan. For quantification of HRP and Coracle labeling intensity in mys mutant class I clones, cumulative average fluorescence intensities were 163 for high Coracle

regions and 82 for low Coracle regions. For quantification of Coracle immunofluorescence intensities in crossing dendrites in Figure 7G, line scans were performed up to the point at which dendrites crossed. Statistical analysis was performed using R (R Development not Core Team). Normality of data sets was assessed using a Shapiro-Wilk test. All p values are indicated as: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Mature third instar larvae were dissected in PBS and fixed immediately with 3% glutaraldehyde in 0.1 M phosphate buffer (PB). Specimens were fixed for a total of 20 min, with 60 s of the fixation time in a Pelco 3451 Microwave System. Fixed tissue was washed 3 × 20 min in 0.1 M PB, postfixed with 1% osmium tetroxide in 0.1 M PB in a microwave for 2 × 40 s (each 40 s exposure in fresh osmium), then washed 3 × 10 min in 0.1 M PB. Tissue was dehydrated in the microwave in ethanol grades of 50%, 70%, 95% (all 1 × 40 s), and 100% (2 × 40 s). Dehydrated tissue was infiltrated in epon (Fullam Epox 812) and ethanol (1:1) for 15 min in the microwave, then in 100% epon resin (2 × 15 min each with fresh epon) in the microwave. Specimens were then mounted between two plastic slides with epon and polymerized overnight at 60°C.