Fitc labelled goat anti rabbit igg

Polyclonal antibodies against recombinant ppFhCL3 and recombinant FhCL3 zymogen were obtained from rabbits immunised with each of the respective recombinant proteins (Eurogentec). F. hepatica NEJs 6 h and 24 h post-excystment were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 1 h at room temperature (RT). After three washes with antibody diluent (AbD: 0.1% [v/v] Triton X-100, 0.1% bovine serum albumin and 0.1% [w/v] sodium azide in 1x PBS), the NEJs were probed with either anti-ppFhCL3, anti-FhCL3 or rabbit pre-immune antiserum diluted 1:500 in AbD buffer, overnight (ON) at 4 °C. After three washes in AbD, the secondary antibody, fluorescein isothiocyanate (FITC)-labelled goat anti-rabbit IgG (Sigma-Aldrich) (1:200), was added and incubated ON at 4 °C. Phalloidin-tetramethylrhodamine isothiocyanate (TRITC) (200 μg/mL [w/v]) (Sigma-Aldrich) was used to counter-stain the muscle tissue and provide structure. All the specimens were whole-mounted in a glycerol solution containing 0.1 M propyl gallate and visualised in a confocal scanning laser microscopy (CSLM) (Leica TCS SP5) under the HCX PL APO CS 100x oil objective lens, using a Leica type F immersion oil.

NEJ immunolocalisation studies were carried out as previously described [12 (link)] using excysted F. hepatica NEJ (Italian isolate; Ridgeway Research Ltd, St Briavels, UK) cultured for 6 h and 24 h that were fixed with 4% paraformaldehyde in 0.1 M PBS (Sigma-Aldrich, St Louis, MO, USA) for 1 h at room temperature. The anti-FhStf-1, anti-FhStf-2, anti-FhStf-3 or rabbit pre-immune antiserum were prepared in rabbits against recombinant FhStf-1, FhStf-2 and FhStf-3 (Eurogentec, Seraing, Belgium) using 200 µg of each antigen per immunisation; four injections in total were given at 0, 2, 4 and 8 weeks of the immunisation protocol. Cross-reactivity between the antibodies was assessed prior to immunolocalisation studies (Figure S1). The antibodies were used at 1:500 dilution. Bound antibody was visualised using a 1:200 dilution of the secondary antibody, fluorescein isothiocyanate (FITC)-labelled goat anti-rabbit IgG (Sigma-Aldrich, St Louis, MO, USA). Images were captured by confocal scanning laser microscopy (CSLM) (Leica TCS SP8, Leica Microsystems, Milton Keynes, UK) under the HCX PL APO CS 100x oil objective lens at room temperature and processed using the Leica Application Suite X (LAS X; v.2.0; Leica Microsystems, Milton Keynes, UK).

Samples were fixed with 4% formalin for 30 min at 4 °C and post-fixed with 70% ethanol for 24 hours at 20 °C; 0.2 ml of suspension containing 20×10⁶ fibroblasts/ml in culture medium were incubated for 1 hour at room temperature with the primary anti-HN antibody (1:200), a rabbit polyclonal antibody raised against HN protein (Thermo Fischer Scientific, Rockford, IL61105, USA). The secondary antibody used was FITC-labelled goat anti-rabbit IgG (1:30) (Sigma-Aldrich Corp., St Louis, MO, USA). Nuclei were counter-stained with 100 ng/ml 4ʹ,6-diamidino-2-phenylindole (DAPI) (Cytocell, Banbury, UK). Slides were observed and cells were visually scored at 200× and 400× magnification. Immunostaining was examined using a fluorescent microscope BX-51 (Olympus,Jappan). Of all cases and controls at least 200 cells were examined. Cells examined were classified with the following scoring: high level positivity ++, slight positivity + -, negative -. Levels ++ and + - were considered positive for statistics.