20 μm polystyrene beads

Embryos were permeabilized by performing perm-1 (RNAi) [62 (link)]. Embryos or oocytes were then dissected into Shelton’s Growth Media (Inulin, 1 mL of 5 mg/mL stock; Polyvinylpyrrolidone powder, 50 mg; BME vitamins, 100 μl of 100x stock; chemically defined lipid concentrate, 100 μl; 100 concentrated Pen-Strep, 100 μl; Drosophila Schneider’s Medium, 9 mL) with 20 μm polystyrene beads (Polysciences, Warrington, PA), and sandwiched between a large and small coverslip sealed on two parallel edges with VALAP (1:1:1, vaseline:lanolin:paraffin wax) as in [63 (link)]. Drug was introduced to the sample through by capillary action by placing a drop of drug-containing solution at one side of the sample, and touching a piece of filter paper at the opposite side.

Transgenic embryos from strain FC254 were obtained by standard dissection from non-Unc gravid adults and were mounted in Egg Buffer (118mM NaCl, 48mM KCl, 3mM CaCl, 3mM MgCl, 5mM Hepes pH7.0) with 1% methyl cellulose (Sigma-Aldrich, #274429) and 0.09% 20-μm polystyrene beads (a 1:30 dilution from stock suspension, Polysciences, Inc., #18329). Time-lapse, widefield fluorescence microscopy (PlanApo 60X, 1.4 NA, Nikon Eclipse TE300 with automatic shutter control, Cooke Sensicam cooled CCD, Metamorph acquisition control software) was used to record the localization of EFF-1(S632/634/654A)::GFP in embryos. Eight one-micron-spaced optical sections through the dorsal or ventral hypodermal surface were imaged every 2.5 minutes for 15 timepoints. Maximum intensity Z-projections of four (ventral) or five sections (dorsal) were rendered using ImageJ [74 ]. FC183 embryos were imaged using the techniques described under “Monitoring Cell-Cell Fusion”.