Fig 1. Liver histopathology.
Panel A: Liver of Delta Smelt from the No Feeding treatment at Day 4. Arrows indicate eosinophilic cytoplasmic inclusion bodies, possibly autophagosomes. Panel B: Liver of Delta Smelt from the No Feeding treatment at Day 7, cytoplasmic inclusion bodies (indicated by arrows) at higher magnification. Note possible double membranes. Panel C: Liver of Delta Smelt from the Feeding treatment, PAS stain. Arrows indicate glycogen granules in the cytoplasm of hepatocytes that are stained magenta (compare to panel D). Panel D: Liver from Delta Smelt from the No Feeding treatment at Day 21, PAS stain. No magenta glycogen granules observed in cytoplasm of hepatocytes (compare to panel C). Panel E: Liver of Delta Smelt from the Feeding treatment, H&E stain; the numbered hepatocytes were used to measure hepatocyte area. Panel F: Liver of Delta Smelt at Day 21 from the No Feeding treatment, H&E stain. Note atrophied hepatocytes (compare to Panel E). For both the Feeding and No Feeding treatments, hepatocytes were selected for area measurement based on three criteria: 1) well demarcated cell membranes, 2) presence of nucleolus in the nucleus, and 3) cells were along a ‘backward S’ path moving from the top-left corner to the bottom-right corner of each photo (Panels E and F). Scale bars = 20 μm (Panel A), 8 μm (Panel B), 15 μm (Panels C and D), 20 μm (Panels E and F).Using the formalin-fixed fish, liver tissue from the No Feeding treatment at Day 0, 21, 42, and 56 was prepared for immunohistochemical detection of apoptosis. Liver tissue from two fed fish was used as a control (Day 0 and 49). Tissues for immunohistochemistry were sectioned (3 μm) and adhered to Superfrost®Plus Microscope Slides (Fisher Scientific, Pittsburgh, PA, USA). Copper-exposed olfactory tissue from Delta Smelt was used as positive control and normal horse serum was substituted for the primary antibody as a no primary antibody control. After deparaffinization and rehydration, heat-induced epitope retrieval was performed in citrate buffer (pH 6) at 95–98°C for 10 min in a microwave (800W). Endogenous peroxidase activity was blocked using 3% H2O2 in methanol for 15 min followed by non-specific blocking with 10% normal horse serum for 15 min. Anti-cleaved caspase-3 polyclonal antibodies (Cat# G7481, Promega, Madison, WI, USA) at 1:2000 dilution were applied to the sections overnight at 4°C and subsequently incubated with HRP-conjugated secondary antibodies for 30 min at room temperature. Finally, antigen-antibody complexes were visualized with chromogen for 1 min. Sections were counterstained with Meyer’s hematoxylin for 15 sec and mounted with Krystalon™ mounting media (MilliporeSigma, Burlington, MA, USA).