The tench were dissected and sexed before the digestive tract from each was removed and opened longitudinally in search of helminths. For tapeworms found still attached to the intestine, their position was registered before a 15 × 15 mm piece of tissue that surrounded the site of attachment was excised and then fixed in either chilled (4°C) bouins or in 10% neutral buffered formalin for 24 h. The bouin
fixed material was subsequently rinsed in several changes of 4°C 70% ethanol before being stored in the same medium until processed for histology. After fixation, the tissues were dehydrated through an alcohol series and then paraffin Metformin supplier wax embedded using a Shandon Citadel 2000 Tissue Processor (Shandon Citadel 2000, London, UK). After blocking out, 5-μm-thick sections were cut and then stained with haematoxylin and eosin and/or alcian BMN 673 purchase blue 8 GX pH 2·5 and periodic acid Schiff’s reagent (AB/PAS). Multiple histological sections were taken from each tissue block, examined and photographed using a Nikon Microscope ECLIPSE 80i (Nikon, Tokyo, Japan). For transmission electron microscopy (TEM), 7 × 7 mm pieces of infected intestinal tissue were fixed in chilled 2·5% glutaraldehyde in
0·1 m sodium cacodylate buffer for 3 h. The fixed tissues were then post-fixed in 1% osmium tetroxide for 2 h and then rinsed and stored in 0·1 m sodium
cacodylate buffer containing Venetoclax in vitro 6% sucrose for 12 h. Thereafter, the pieces of tissue were dehydrated through a graded acetone series and embedded in epoxy resin (Durcupan ACM, Fluka). Semi-thin sections (1·5 μm) were cut on a Reichert Om U 2 ultra microtome and stained with toluidine blue. Ultra-thin sections (90 nm) were stained with 4% uranyl acetate solution in 50% ethanol and Reynold’s lead citrate and then examined using an Hitachi H-800 transmission electron microscope (Hitachi H-800, Tokyo, Japan). For each method, corresponding pieces of uninfected intestine were also processed, so that a direct comparison with the infected material could be made. For comparative purposes, the number of granulocytes in an area measuring 30 000 μm2 was determined using a Nikon Microscope ECLIPSE 80i and computerized image analysis software (Nis Elements AR 3.0) in 10 separate zones on each section of infected fish (i.e. in the submucosa layer close to the site of cestode attachment) and in 10 separate areas on each section of uninfected fish material. Granulocyte subsets (i.e. neutrophils and mast cells) were identified on subcellular features observed using transmission electron microscopy.