Liver Immunohistochemical Analysis of LC3 and p62

The immunohistochemical analysis was performed as described previously [14 (link), 15 (link)], using formalin-fixed, paraffin-embedded liver sections. After deparaffinization and rehydration in xylene and graded alcohols, endogenous peroxidase was quenched with hydrogen peroxide. Nonspecific binding was blocked with 10% normal goat serum in phosphate buffered saline (PBS) (Wako, Tokyo, Japan). Incubation with anti-LC3 rat antibody (dilution 1:200, Cell Signaling Technology, Denver, MA, USA) and anti-p62 antibody (dilution 1:1000, MBL, Nagoya, Japan) were followed by incubation with goat anti-rabbit IgG secondary antibody (dilution 1:1000, Thermo Fisher Scientific, Waltham, MA, USA). Specimens were observed under a microscope (Keyence BZ-9000, Osaka, Japan). For the semi-quantitative morphometric analysis, the numbers of LC3 and p62 positive cells were calculated in a blinded fashion and averaged for five fields per slide at a 200X magnification by using Image-Pro Plus computerized image analysis system version 4.5 (Media Cybernetics, Silver Spring, MD, USA).