Wild-type (WT) lines (TAB5 and TAB14) and mutant lines (alleles foigrhi1532b and mbtps1hi1487) Selleckchem HDAC inhibitor were maintained in accordance with the policies of the institutional
animal care and use committee of the Mount Sinai School of Medicine. Mutants were genotyped as described.21Tg(fabp10:RFP;ela:GFP) fish were obtained from D. Stainier (University of California at San Francisco). Morpholinos targeting the anti-thymocyte globulin initiator of atf6 (gene name si:ch211-199m3.9; 5′-ACATTAAATTCGACGACATTGTGCC-3′) or sterol regulatory element binding protein cleavage-activating protein (scap)22 and a nontargeting control (5′-CCT CTTACCTCAGTTACAATTTATA-3′) were ordered from Gene Tools, LLC (Philomath, OR). The morpholinos were diluted in water to a 0.5 mM stock, and approximately 5 pmol was injected into the early embryos. The tunicamycin (TN) treatment protocols are detailed in the Results section. Whole-mount Oil Red O staining was carried out as described.22 Steatosis was scored in larvae with three or more lipid droplets in the liver parenchyma. A Nikon SMZ1500 equipped with a Nikon DS-2M color camera was used to acquire images, which were edited with Photoshop. The amount of Oil Red BAY 73-4506 in vivo O staining per liver cell was quantified with
Metamorph software (Molecular Devices) on cryosections stained with Oil Red O and 4′,6-diamidino-2-phenylindole (DAPI). In each bright field image, a region outlining the liver was selected, and Oil Red O–stained particles were selected by color thresholding and were
counted. The total area occupied by Oil Red O staining was measured. Each measurement was divided by the number of DAPI-stained nuclei within the region. At least five sections per fish were measured for at least three fish per group. At least four WT and foigr mutant larvae fixed in 4% paraformaldehyde were embedded in plastic as described.23 Four-micrometer sections were incubated in 0.5% periodic acid, washed, stained with Schiff’s reagent (5 g/L basic fuchsin, 0.1 N hydrochloric acid, and 0.045 potassium check details metabisulfite), washed with running tap water, and counterstained with hematoxylin. Images were taken with an Olympus BX41 microscope and a Nikon DS-Ri1 color camera. Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed with a Roche in situ cell death detection kit as described.24 Hepatocytes were stained with cyanine 3/streptavidin (1:200; Sigma), and nuclei were labeled with DAPI. The percentage of apoptotic hepatocytes was calculated for at least 15 sections (which represented at least three fish per group) through the division of the number of TUNEL-positive hepatocytes by the total number of nuclei in each section.