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.