2.5. Staining for calcium phosphate deposits
Sea bream larvae with large (N50 μm) calculi in their urinary bladder
were collected and preserved in 10% neutral buffered formalin until
they were processed for histology. Preserved larvae were dehydrated,
paraffin wax embedded and sectioned (5 μm) (Leica RM2245,
Germany). Slides containing whole larvae sections were first stained
with hematoxylin and eosin before proceeding to calcium phosphate
staining. Staining for calcium phosphate deposits was carried out
using 2% Alizarin Red S (Sigma-Aldrich, USA) in distilled water adjusted
to a pH of 4.2. Slides were stained for approx. 2 min then dehydrated in
acetone, dipped in an acetone-xylene (1:1) solution and cleared in
xylene before mounting.