Histologic and immunohistochemical examinations
For immunofluorescence staining of 3D liver spheroids, spheroids were fixed for 30 min in 4% w/v paraformaldehyde in PBS. Then whole samples were permeabilized with 0.1% v/v Triton X-100 in PBS for 5 min and pre-blocked for 30 min with 10% FBS diluted in PBS. Next, spheroids were incubated with mouse glial fibrillary acidic protein (GFAP; 1:50, Developmental Studies Hybridoma Bank (DSHB), cat # 8-1E7) for HSCs, rabbit CYP3A4 antibody (1:200, Fisher Scientific, cat# 50-173-2058) for HepG2 cells, anti-human E-cadherin monoclonal antibody (1:200, R&D, cat# MAB1838) and anti-vinculin antibody (1:200, Santa Cruz, cat# sc-25336) overnight in the fridge. The next day we stained the spheroids with Alexa Fluor 546 goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody (Thermofisher, cat# A11030) and Alexa Fluor 546 donkey anti Sheep IgG (H+L) Secondary Antibody (Thermofisher, cat # A21098) for 90 min, as appropriate. The spheroids were then incubated with 10 µM Hoechst 33342 (Thermofisher, cat# H3570) diluted in 1% BSA for nuclei detection for 5 min before collecting confocal microscope images (Nikon Corporation confocal microscope, inverted). The step size of Z-stacks was set to 10 µm thickness and 30-40 slices per image were taken to image entire spheroids.