D5 fi1

F₁ livers from three fish from each parental groups were dissected at 142 DPF, rinsed in 1x PBS and fixed in 4% paraformaldehyde overnight at 4 °C, followed by dehydration, infiltration and embedding in Technovit 7100 following the manufacturers’ protocol (Kulzer Histo-Technik). Semi-thin sections (1 µm) were cut on a microtome (Leica, model #RM2155) and stained with toluidine blue. Sections were imaged on an Olympus BX51 microscope attached to a Nikon D5-Fi1 camera. Quantification of stained liver tissue was performed using ImageJ (Rasband, 1997–2015). Images were converted to RGB stacks (selected image/type/RGB stack, followed by Image/stacks/make montage) and the red channel used to segment the image into stained vs non-stained tissue. Toluidine blue is a metachromatic dye that stains proteins, nucleic acids and membranes. Non-stained tissues are lipids, solutes and the lumen of the blood vessels, bile canaliculi and sinusoids. The images were converted to binary images (image/adjust/threshold) and the percentage of stained vs non-stained tissue was measured from each fish and used as an approximate for liver lipid content.