Histological Analysis of Liver Tissues

Liver tissues were fixed in Bouin's solution (Sigma, St. Louis, USA), embedded in paraffin, sectioned at 6 μm, and stained using hematoxylin and eosin (H&E; Sigma, St. Louis, USA) and Masson's trichrome stain (Trichome stain kit; ScyTek Laboratories, West Logan, USA) using standard protocols [20 (link), 21 (link)]. Nuclear counterstaining was performed using hematoxylin, and the samples were examined using light microscopy (Nikon, Tokyo, Japan). For Slc25a15 immunostaining, the samples were incubated first with 1 : 300 dilutions of the anti-Slc25a15 antibody (Abcam, Cambridge, UK) and then with a biotinylated anti-mouse IgG (Vectastain ABC Kit; Vector Labs, Burlingame, USA). The sections were incubated with the avidin–biotin–peroxidase complex (Vectastain ABC Kit; Vector Labs, Burlingame, USA) and DAB. The Celleste image analysis software (Thermo Fisher Scientific, Waltham, USA) was used to count the number of immunoreactive cells.