Fillets of torafugu and red seabream (500–750 g) were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde (PFA) at 4 "C for 1 week (n = 3), and stained with 0.3% oil red O in a mixture of isopropanol and distilled water (6:4 v/v). The fillets were then washed with distilled water and their images captured using a digital camera.
For the oil red O staining of the frozen sections, skeletal muscle, liver, skin, intestine, and gills of torafugu and red seabream (n = 2 and 3, respectively) were fixed in 4% PFA/PBS at 4 "C overnight. Tissues were washed twice with 0.2 M phosphate buffer for 30 min. Subsequently, the tissues were sequentially treated with 0.2 M phosphate buffer solutions containing 10%, 20%, and 30% sucrose, for 30 min each, and frozen-sectioned at a thickness of 16 lm. The sections were briefly washed in distilled water, immersed in 60% isopropanol for 1 min, and stained for 15 min with the oil
red O solution. The sections were lightly counterstained with
hematoxylin. Images were captured using a MVX10 microscope
(Olympus, Tokyo, Japan).