Flip Your Own PTEN In To A Total Goldmine

The size of the peroneal nerve was quantified in whole-mount embryo preparations by manually buy PD0332991 outlining the nerve, visualized through Hb9::GFP fluorescence, and measuring GFP signal intensity. To account for embryo-to-embryo variability in Hb9::GFP levels, the fluorescence of a characteristic ectopic site of Hb9::GFP expression was used to normalize nerve-associated fluorescence in each embryo. A semi-automated quantification of axon outgrowth from motor explants was performed using the FeatureJ plugin of NIH ImageJ (Grider et?al., 2006) that uses a Hessian-based algorithm to detect linear structures, such as axons. The algorithm was applied to images of Hb9::GFP fluorescent axons after removal of the signal associated with the motor columns to measure the total axonal area (#pixels). The percentage of growth cone collapse was quantified as previously described (Gallarda et?al., 2008). Pearson's correlation coefficients were calculated using the Coloc_2 plugin of NIH ImageJ/Fiji. Quantification of immunoblots was performed with NIH ImageJ. The unpaired t test (two-tailed), Dunnett's multiple comparison test (after one-way ANOVA), and Tukey's multiple comparison test (after one-way ANOVA) were performed with GraphPad Prism software. The Kolmogorov-Smirnov test was performed with IGOR Pro software. p?PTEN of whole-mount neurofilament staining. Representative images of the peroneal nerve of Hb9::Gfra1 and control embryos were acquired with a Praire Technologies TSA HDAC supplier Ultima two-photon microscope. Images were acquired with 4x (for whole-mount preparations), 10x, 20x, 60x objectives on an Olympus Fluoview 1000 confocal microscope. For the Dunn's chamber assay, a long distance 20x objective was used. With the exception of surface staining and PLA detection on growth cones, which were acquired as single confocal sections, all other images correspond to maximal Z projections of confocal stacks. To obtain the whole motor projection pattern at the sciatic plexus, confocal stacks acquired from adjacent transverse sections were combined using the Mosaic plugin of NIH ImageJ. We thank Laura Franco for technical assistance; Christopher Hinckley for help with two-photon imaging; Shane Andrews for help with microscopy; Shawn Driscoll for help with data analysis; Arnon Rosenthal and Buffer Fennie for Gfra1 mice; Bennett Novitch for Olig2::Cre mice; Kuo-Fen Lee for p75 mice; Martin Goulding and Andrew Boyd for EphA4 mice; Frank Costantini for Ret mice; Tsung-Chang Sung, Zhijiang Chen, Kuo-Fen Lee, Yoo-shick Lim, and Dennis O'Leary for ephrin-A2/5 and p75 plasmids and helpful discussion. D.B. was supported by HHMI, O.C. by an NINDS fellowship, G.B. by HHMI and a Salk pioneer fellowship, and S.L.P. is an investigator with HHMI.