Alexa fluor 488donkey anti

Confocal microscopy was carried out as described (27 (link), 28 (link), 29 (link)). Huh7 cells were seeded onto coverslips (1.0 × 10⁵ cells/ml). About 48 h later, cells were fixed and permeabilized with cold methanol for 10 min at −20°C. The cells were then incubated with a goat anti-apoB polyclonal antibody and rabbit anti-Surf4 antibody overnight at 4°C. Antibody binding was detected using Alexa Fluor 647 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-goat IgG. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Thermo Fisher). After washing, coverslips were mounted on the slides with ProLong Diamond Antifade Mountant (Thermo Fisher). Localizations of apoB and Surf4 were determined using a Leica SP5 laser scanning confocal microscope (filters: 461 nm for 4′,6-diamidino-2-phenylindole, 519 nm for Fluor 488, and 633 nm for Fluor 647).