Liver samples were fixed overnight in 4% paraformaldehyde for cryosections, or 10% neutral buffered formalin for paraffin embedding. Endogenous biotin-blocked, 4-µm-thick paraffin sections were stained with haematoxylin, anti-C3d antibody (R&D Systems, Inc.) or anti-F4/80 antibody (Biolegend, Inc.)37 (link),38 (link). Six-µm-thick cryosections were stained with Oil Red O (Sigma-Aldrich) and Mayer's Hematoxylin Solution (Fujifilm Wako Pure Chemical Corporation)39 (link). Captured images were analysed using the BZ-X800 Analyzer (Keyence Corporation) or ImageJ (Fiji)40 (link). C3d-positive area and F4/80-positive cells per hepatocyte were calculated.
Free full text: Click here
