Cellssens software

Eyes were harvested from fish not used in the experiments to measure retinal responses to light stimuli. Food was withheld for 24 hours and 10–12 fish from each diet were anesthetized in a bath containing 25 mg L^-1 MS-222 (Syndel, Ferndale, WA, USA) buffered with 50 mg L^-1 sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA). Upon removal from the anesthesia bath, the spinal cord was immediately severed, and eyes harvested. Retinal tissue was subsequently embedded, sectioned, mounted on glass slides, and stained with Hematoxylin-Eosin using standard histological procedures. Images were captured using a BX53 microscope outfitted with a DP73 camera and visualized with the cellsSens software (version 510, Olympus, Center Valley, PA, USA).

Thoracic aortae (n = 6–7 per group) were fixed in 1% neutral buffered formalin for 24 h at room temperature and dehydrated in an increasing series of ethanol prior to being embedded in paraffin blocks. The sections of aortae were cut at 5 μm. Mounted sections were blocked in 10% goat serum at room temperature for 20 min then washed with 10 mM PBS for 5 min. Sections were incubated with primary antibody anti‐rabbit TLR4 (Novis Biologicals; NB100‐56580; 1:500 dilution in PBS) at 4°C overnight and subsequently washed with 10 mM PBS for 5 min and incubated with Alexa Fluora 594 goat anti‐rabbit IgG (Molecular Probes; A11012; 1:2000 in PBS) at room temperature for 1 h. After washing slides with 10 mM PBS for 10 min (three times), slides were mounted with ProLong Diamond Antifade Mountant with DAPI (Invitrogen). An investigator blinded to the group assignments captured representative images of the aortic endothelium (at least 10 regions of interest per aorta) using CellsSens Software (Olympus, Center Valley, PA). The total integrated intensity was measured within the region of interest and divided by the total area (ImageJ v1.8). These values were averaged for each sample.