Tecnai 12 spirit tem

Adult female B. pahangi worms were incubated with either 1 μM, 0.3 μM, or 0.1 μM auranofin, 10 μM flubendazole (as a positive control [28 (link)]), or 1% DMSO overnight, then cut into 3 segments separating the anterior, middle and posterior sections. The middle sections were further cut into 1 mm sized pieces in fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.3–7.4) and stored at 4°C. Middle sections were subsequently treated with 1% tannic acid for 1 hour, followed by three buffer washes before post fixation staining with 2% osmium tetroxide for 1 hour. The samples were washed three times in buffer before dehydration in an ethanol series. Worm sections were then infiltrated with propylene oxide, embedded in epon 812 resin and polymerized in a vacuum oven at 60°C overnight. Ultrathin sections were cut using an RMC MTX ultramicrotome with a Diatome diamond knife followed by post staining of the grids with saturated ethanolic uranyl acetate and Reynolds lead citrate. Samples were imaged on a FEI Tecnai 12 spirit TEM operated at 80 kV. A similar procedure was performed on adult female O. ochengi worm masses that were cultured for 7 days with 10 μM auranofin before fixation of cut pieces of the adult female mass. Untreated adult female masses cultured for 7 days and fixed by the same procedure served as the control.

Cells were grown in six‐well plates, three control wells were treated with oleic acid for 24 h and three treatment wells were treated with oleic acid for 24 h plus 0.5 mM caffeine for 6 h. Cells were harvested and fixed in a modified Karnovsky's fixative (2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M Sorenson's phosphate buffer, pH 7.4) overnight at room temperature. Cells were rinsed in Sorenson's phosphate buffer, followed by secondary fixation in 1.0% osmium tetroxide with 0.8% potassium ferricyanide in 0.2 M cacodylate buffer, pH 7.4. Samples were rinsed and dehydrated with an ethanol series, and embedded in Spurr's epoxy resin (Electron Microscopy Sciences). Embedded samples were trimmed, and 90 nm sections were cut on a Leica UC7 ultramicrotome (Leica Microsystems, Inc.). Sections were collected on Formvar‐coated (0.25 g Formvar in 100 ml ethylene dichloride), 200 mesh copper grids. Sections were stained with 2% uranyl acetate in 50% ethyl alcohol for 15 min, rinsed with DI, and stained with Reynolds lead citrate for 15 min. Sections were imaged on a Tecnai 12 Spirit TEM (FEI). A total of 30 images were collected for each treatment group and images were analyzed in Fiji (Schindelin et al., 2012). Point counting stereological analysis was used to determine volume density of autophagosomes and mitochondrial.