Goat anti mouse albumin

After 1 day and again after 5 days, PBS-rinsed primary hepatocytes in various systems were fixed with 8% formaldehyde for 20 minutes and were made permeable with 0.2% Triton X-100 (Nacalai Tesque) for 5 minutes. After blocking with Blocking One solution for 1 hour, the cells were incubated with primary antibodies to albumin (goat anti-mouse albumin; Abcam, Cambridge, UK) or ASGPR (rabbit anti-human ASGPR1/2; Santa Cruz Biotechnology, Dallas, TX, USA) for 2 hours at room temperature. The cells were triple-rinsed with PBS and incubated with the corresponding secondary antibodies (Alexa 555-conjugated donkey anti-goat IgG, Thermo Fisher Scientific; Alexa 488-conjugated goat anti-rabbit IgG, Thermo Fisher Scientific) for 2 hours; the cells were then counterstained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, St Louis, MO, USA) to label the cell nuclei. After rinses with PBS, protein staining in the cells was examined with a fluorescent inverted microscope (Olympus Corporation, Tokyo, Japan).