Staining for FhGST-S1 was performed on formalin-fixed and paraffin-embedded tissue sections according to the method described previously [45] . Sections were washed in Tris-buffered saline (TBS; 0.1 M Tris-HCl with 0.9% NaCl [pH 7.2]), treated with 0.05% (w/v) protease (type XXIV, bacterial: Sigma) in TBS for 5 min at 37uC for antigen retrieval, before three further 5 min washes in ice-cold TBS. Following TBS washes, sections were incubated for 10 min in 50% (v/v) swine serum in TBS followed by incubation for 15-18 h at 4uC in rFhGST-S1 polyclonal antibody (diluted at 1:500 in 20% swine serum in TBS). Sections were again washed in TBS before further incubation at ambient temperature (approximately 20uC+/23uC) with anti-rabbit peroxidise anti-peroxidase (PAP; diluted at 1:100 in 20% swine serum in TBS). Following washes with TBS, sections were incubated, with stirring, for 10 min, with 3,3-diaminobenzidine tetrahydrochloride (DAB; Fluka, Buchs, Switzerland) with 0.01% v/v hydrogen peroxide in 0.1 M imidazole buffer pH 7.1, before counterstaining with Papanicolaou's hematoxylin for 30 s. Sections were then rinsed, dehydrated in alcohol, cleared in xylene, and mounted. Consecutive sections from each tissue were used as negative controls in which the rFhGST-S1 polyclonal antibody was replaced by TBS.