H 600 electron microscope

Worms were harvested from infected mice and treated with PZQ as described for movement assays, then fixed overnight at 4°C in 2.5% glutaraldehyde/2% paraformaldehyde in 0.1M sodium cacodylate (pH 7.3). Worms were then washed in 0.1M sodium cacodylate (3x10 minutes) and post-fixed on ice (2 hours) in reduced 1% osmium tetroxide. Worms were washed in distilled water (2×10 minutes), stained in alcoholic uranyl acetate (overnight, 4°C), rinsed in distilled water, dehydrated in MeOH (50%, 75% and 95%), followed by successive rinses (10 minutes) in 100% MeOH and acetonitrile. Worms were incubated in a 1:1 mixture of acetonitrile and epoxy resin for 1 hour prior to 2×1-hour incubations in epoxy resin, then cut transversely and embedded overnight in epoxy resin (60°C). Ultra-thin sections (70nm) were cut onto bare 200-mesh copper grids, stained in aqueous lead citrate (1 minute), then imaged on a Hitachi H-600 electron microscope fitted with a Hamamatsu C4742-95 digital camera operating at an accelerating voltage of 75 kV.

Adult worms were harvested and recovered as above. Fixation was carried out overnight at 4°C in 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M sodium cacodylate (pH 7.3). Worms were washed 3 × 10 minutes in 0.1 M sodium cacodylate and post-fixed for 2 hours on ice in reduced 1% osmium tetraoxide. Worms were then washed 2 × 10 minutes in distilled water and stained overnight at 4°C in alcoholic Uranyl Acetate. Worms were rinsed in distilled water, dehydrated in 50%, 75% and 95% MeOH, followed by successive 10 minute rinses in 100% MeOH and acetonitrile. Worms were incubated in a 1:1 mix of acetonitrile and epoxy resin for 1 hour prior to 2 × 1 hour incubations in epoxy resin. Worms were then cut transversely and embedded overnight in epoxy resin (60°C). Ultra-thin sections (70 nm) were cut onto bare 200-mesh copper grids and stained in aqueous lead citrate for 1 minute. Sections were imaged on a Hitachi H-600 electron microscope fitted with a Hamamatsu C4742-95 digital camera) operating at an accelerating voltage of 75 kV.