Jb 4 resin

Adult F. hepatica were collected from the bile ducts of sheep (as described above) and immediately placed in PBS containing 4% paraformaldehyde (Sigma-Aldrich) for 4 h at room temperature. Flukes were subsequently embedded in JB-4 resin (Sigma-Aldrich) according to the manufacturer’s instructions. Semi-thin (2 µm) sections were cut on a pyramitome and mounted on clean glass slides and stained with 1% (w/v) toluidine blue for 30 s or subjected to immunofluorescent labelling according to Bennett et al. [16 (link)]. Briefly, tissue sections were washed in PBS then incubated overnight in primary antibody in antibody diluent (AbD: PBS containing 0.2% (v/v) Triton X-100) at room temperature. The anti-tyrosinated α-tubulin mouse monoclonal (TUB-1A2; Sigma-Aldrich) was used at a 1:100 dilution whilst the rabbit polyclonal antibody (Genscript) raised against FhRal-A was used at 10 µg/mL. As a negative control, tissue sections were also incubated with pre-immune serum in AbD. The sections were then washed three times in AbD before incubation in an appropriate secondary antibody-FITC/-TRITC conjugate at a 1:200 dilution in AbD for 1 h at room temperature. The sections were counterstained in 300 nM DAPI (ThermoFisher) and mounted in glycerol containing 10% (v/v) PBS and 0.1 M propyl gallate (Sigma-Aldrich). Sections were viewed under a Leica DM2500 fluorescent microscope.

Adult flukes were fixed in paraformaldehyde at 4 °C overnight, washed with PBS, dehydrated with ethanol and embedded in JB-4 resin (Sigma-Aldrich)^{4 (link),10 (link)}. Sections (2 μm) were incubated in either an anti-peptide antibody raised in mice to the following FhKT1.1 amino acid sequence, Cys-Glu-Gly-Asn-Asp-Asn-Arg-Phe-Asp-Ser-Lys-Ser-Ser-Cys, or pre-immune sera, each at a 1:500 dilution. Sections were then washed three times in PBS with 0.5% Triton X-100 for 30 min and incubated in a 1:500 dilution of the secondary antibody, fluorescein isothiocyanate (FITC)-labelled goat anti-mouse immunoglobulin (Sigma-Aldrich). After three washes in PBS with 0.5% Triton X-100 for 30 min sections were dried and coverslips mounted using glycerol:PBS (9:1) containing 100 mM propyl gallate. Sections were viewed using a Leica DM 2500 light microscope under the HCX PL FLUOSTAR × 10 and × 40 lenses.