Point scanning confocal microscope

Wandering late third-instar larvae were dissected for NMJ phenotype analysis. Briefly, the tissue was fixed with 8% formaldehyde in phosphate-buffered saline (PBS) for 10 min, washed with PBT (PBS containing 0.1% Triton X-100), and blocked with 5% normal donkey serum in PBT for 1 hour at room temperate. The larvae were incubated with Dlg1 antibody overnight at 4°C, followed by incubation with Alexa Fluor 488–conjugated secondary antibody against mouse IgG and Alexa Fluor 568–conjugated phalloidin. Confocal images were acquired using a point scanning confocal microscope (Leica). Images for quantification of NMJ bouton number on muscles 6 and 7 at segment 3 were produced by projection of z-sections using the ImageJ software blindly.

Eight-well chamber slides (Nunc Lab-Tek) were coated with poly-D-lysine (0.1 mg/ml) overnight, followed by laminin (20 µg/ml) for 1 hour at 37°C. N1E cells (50,000) were seeded onto each chamber, transfected or infected with siRNAs or adenovirus vectors, and treated with BMP7 with or without inhibitors [LIMKi-3 (Millipore) and LDN] as indicated in the figure legends. The cells were fixed, permeabilized, stained with Alexa Fluor 568–conjugated phalloidin (Life Technologies), as described in the manufacturer’s protocols, and observed under a point scanning confocal microscope (Leica). The phalloidin signal on filopodia was traced and quantified using the ImageJ program by a blinded investigator.

Wandering late third instar larvae were dissected for analysis of NMJ phenotype. After dissection, the tissues were fixed with 8% formaldehyde in PBS for 10 min, washed with PBT (PBS containing 0.1% Triton X-100), and blocked with 5% normal donkey serum in PBT for 1 hour at room temperate. The larvae were incubated with DLG (4F3, Developmental Studies Hybridoma Bank) antibody overnight at 4°C, followed by incubation with Alexa Fluor 488– or Alexa Fluor 555–conjugated secondary antibodies against mouse Ig, Alexa Fluor 488–conjugated HRP antibody (123-545-021, Jackson ImmunoResearch), and Alexa Fluor 568–conjugated (A12380, Life Technologies) or Alexa Fluor 647–conjugated (#8940, Cell Signaling) phalloidin. Confocal images were acquired using a point scanning confocal microscope (Leica). Images used for quantification of NMJ bouton number and branch number were derived from a projection of z sections. For quantification of the branch number, branches with at least three boutons were counted blindly.