*2.8. Immuno-Detection of Native FhSOD1 and FhSOD3 in NEJ, Adult Parasites and Their Extracts*

Localisation of FhSOD1 and FhSOD3 in NEJ was conducted as follows: *F. hepatica* metacercariae (Italian isolate; Ridgeway Research, St. Briavels, UK) were excysted and cultured in RPMI 1640 medium containing 2 mM L-glutamine, 30 mM HEPES, 0.1% (*w*/*v*) glucose, 2.5 μg/mL gentamycin, and 10% foetal calf serum (ThermoFisher Scientific) for 24 h, as previously described [29]. NEJ were then fixed with 4% paraformaldehyde (PFA) in 0.1 M PBS (Sigma-Aldrich) pH 7.4, for 1 h at RT. After three washes in antibody diluent (PBS containing 0.1% (*v*/*v*) Triton X-100, 0.1% (*w*/*v*) bovine serum albumin and 0.1% (*w*/*v*) sodium azide; AbD buffer), the NEJ were incubated in rabbit pre-immune sera, anti-rFhSOD1, anti-rFhSOD3, or anti-rFhCL3 polyclonal antibodies (used as a non-related positive control) diluted 1:500 in AbD buffer overnight at 4 ◦C. After three washes in AbD, the NEJ were incubated in a 1:200 dilution of the secondary antibody, fluorescein isothiocyanate (FITC)-labelled goat-anti-rabbit IgG (Sigma-Aldrich) overnight at 4 ◦C in the dark. To counter-stain muscle tissue, the samples were incubated in AbD containing phalloidin-tetramethylrhodamine isothiocyanate (TRITC) (200 μg/mL) overnight in the dark at 4 ◦C. The NEJ were then whole-mounted onto slides using 10% glycerol solution containing 0.1 M propyl gallate and visualised under an Olympus Fluoview 3000 laser scanning confocal microscope using a PL APO CS 6 × 0 oil objective lens. Olympus type F immersion oil was used in viewing and all images were taken at room temperature.

Localisation of FhSOD1 and FhSOD3 in adult *F. hepatica* was conducted on parasites collected from the livers of infected sheep during post-mortem surveillance in Roscommon, Ireland. After collection, the parasites were washed in 1 × PBS and fixed in 4% PFA for 4 h at RT. After 4 h, the PFA was removed, replaced with 1 × PBS followed by an incubation for 1 h at RT. This washing process was repeated twice before dehydration in ascending ethanol and subsequent infiltration with JB-4 resin (Sigma-Aldrich EM0100). Serial 0.5 μM sections were mounted onto slides and probed with rabbit pre-immune sera, anti-rFhSOD1, anti-rFhSOD3, or anti-rFhCL1 polyclonal antibodies (used as a non-related positive control) diluted 1:1000 in PBST for 5 h at RT in a humid container. The sections were washed three times in PBST before addition of a 1:1000 dilution of the secondary antibody, FITC-labelled goat-anti-rabbit IgG (Sigma-Aldrich) in PBST, after which they were placed in a humid container and allowed to incubate overnight in the dark at 4 ◦C. The slides were once again washed three times in PBST and dried, and cover slips were mounted using 10% glycerol solution containing 0.1 M propyl gallate. The sections were visualised under a Leica DM2500 LED optical fluorescent microscope (Leica Microsystems, Wetzlar, Germany).

Adult *F. hepatica* E/S and somatic extract (10 μg/lane) were resolved by gel electrophoresis in 4–20% SDS-PAGE gels and electro-transferred onto nitrocellulose membranes prior to incubation in blocking buffer for 1 h at RT. The membranes were probed overnight at 4 ◦C with rabbit pre-immune sera, anti-rFhSOD1, or anti-rFhSOD3 at a 1:1000 dilution. After washing, the membranes were further probed with the secondary antibody alkaline phosphatase conjugated goat-anti-rabbit IgG at a 1:10,000 dilution for 1 h at RT. Following

final washes, the immune-reactive bands were visualised using the substrate SigmaFast BCIP/NBT (Sigma-Aldrich).
