Jung histoembedder

Mice were anesthetized with halothane and euthanized by decapitation on day 7 after the first ddC administration. The spinal cords were extracted as previously described [57 (link)] and fixed by 4% paraformaldehyde (PFA) in 1× PBS (pH 7.4) at 4 °C for 2 h. Then, the spinal cords were transferred to 25% sucrose at 4 °C overnight. The tissues were stored in 25% sucrose with a few droplets of 10% sodium azide [57 (link)]. On the second day, the spinal cords were trimmed using a dissecting blade to isolate the lumbar (L1–L6) enlargement, placed in cassettes, and processed using an automated tissue processor overnight (ATP 1000 Automatic Tissue Processor). The next day, the tissues were embedded in paraffin (Jung Histoembedder, Leica, Wetzlar, Germany) according to standard protocols. The paraffin blocks were sectioned coronally (5 μm thickness) using microtome machine (Jung Histocut, Leica) and mounted on bond plus slides (Leica, Wetzlar, Germany) for immunofluorescence.

At the beginning and at the end of the experiment 10 pikeperch larvae from each tank were collected and fixed in 10% buffered formalin. For histological process larvae were dehydrated through graded alcohols (70–96°) using a Histokinette 2000 tissue processor (Leica, Nussloch, Germany), then xylene and finally embedded in paraffin wax (Jung Histoembedder, Leica, Nussloch, Germany). Paraffin blocks were sectioned at 5 μm, on a microtome (Leica, RM2135, Leica Instruments, Nussloch, Germany) and stained with hematoxylin, eosin, safran (HES) and examined using light microscopy using a Olympus CX41 binocular microscope (Olympus, Hamburg, Germany), in a range of magnifications (10–40x), connected to an Olympus XC30 camera (Olympus, Hamburg, Germany), which was linked to a computer using image capturing software (CellB®, Olympus, Hamburg, Germany). To assess hepatocyte vacuolization and presence of goblet cells, a three-point scoring system was used (Supplementary Fig. S1), based on a modified method from Betancor et al.^{125 (link)}:

Score 1: no hepatocyte vacuolization or presence of goblet cells.

Score 2: mild hepatic vacuolization or presence of goblet cells.

Score 3: severe hepatocyte vacuolization or presence of goblet cells.