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.