Pioloform covered slotted grids

Approximately, 5 adult hermaphrodites were loaded at room temperature into a 100 μm specimen chamber containing space-filling bacteria and M9 buffer. These worms were frozen instantaneously at ∼ −180°C in either a Leica EM PACT2 (Leica, Germany) or a BAL-TEC HPM010 (Bal-Tec, Liechtenstein) system. The frozen worms were fixed in a Leica EM AFS2 machine using 1% osmium in 0.1% UA in acetone fixative and then embedded in Eponate 12 from Ted Pella, Inc (Redding, CA). Serial sections were cut at a thickness of 40 nm, collected on pioloform covered slotted grids (notchnum 1 × 2 mm oval) from Ted Pella, Inc., and counterstained in 6% aqueous uranyl acetate for 1.5 hr, followed by Reynolds lead citrate for 7 min. Images were obtained on a JEOL JEM 1400 transmission electron microscope (JEOL, Japan) operating at 120 KV. Micrographs were collected using the Gatan Ultrascan 1000XP, 2k × 2k high-resolution camera (Pleasanton, CA). Synapse profiles were used to count the number of synaptic vesicles (∼30 nm in diameter). Each profile represents a single section that passes through the dense projection. Vesicle counting was performed blindly. p values were generated using one-way ANOVA followed by Dunnett's test.