The microinjection of fluorescein-labeled MSP into aged pezo-1 CΔ

The microinjection of 101.6 μM NHS-Fluorescein-labeled MSP-142 into both aged (day 2, 48 hr post mid-L4) wildtype and pezo-1 CΔ hermaphrodites was performed as previously described (Miller, 2001). The injected worms recovered for 4 hr on MYOB plates with OP50 food before imaging. The acquisition of GFP and DIC images was performed by our confocal imaging system (see below) with 1–2 μm z-step size and 10–15 z planes. Time interval for ovulation imaging was every 45–60 s, and duration of imaging was 60–90 min. Ovulation rate = number of successfully ovulated oocytes / total duration of imaging.