Histological diagnostics
The remaining bodies of each clam were dissected and fixed with 5 ml of Davidson fixative (formaldehyde, acetic acid, ethanol) that was replaced with 4% phosphate-buffered formaldehyde after 48 hours. Fixed tissues were then dehydrated in increasing concentration of alcoholic solutions, cleaned in xylenes, and embedded in histological grade paraffin wax. Histological blocks were cut into 3 µm sections on a microtome (RM2245, Leica) and transferred onto microscopic slides that were later dried in 60°C for paraffin dissolution. Prepared sections were dyed using standard Harris’ haematoxylin and alcoholic eosin stain (H&E) protocol for histological visualisation, mounted with xylene-based medium and investigated under light microscope. Only clams with infiltration of NCs observed in more than one tissue type, and prevalence of cancerous cells >50% were classified as DN-positive (Fig. 2.2 ). Diagnosis resulted in selection of 4 neoplastic (labelled as #143, 144, 157, 158) and 2 healthy (#88, 89) individuals as references. Haemolymph and muscle samples from selected individuals were further used for molecular analysis.