Anti hnf4a

Immunofluorescence staining of organoids was performed essentially as described previously^{25 (link)}. Briefly, organoids were fixed with 4% PFA for 2 hours at RT and washed twice with PBS. Organoids were blocked and permeabilized with 5% BSA-PBS including 0.2% Triton-X for 1 hour at RT. Organoids were washed once with PBS and incubated with primary antibody diluted in 2.5% BSA-PBS O/N at 4 ºC. Primary antibodies included anti-AFP (Thermo Fisher, PA5-16658, 1:250); anti-ALB (Bethyl, A80-229A, 1:500); anti-Ki-67 (Thermo Fisher, 14-5698-82, 1:1000); anti-CK7 (Thermo Fisher, MA5-11986, 1:400); anti-CK19 (Cell Signaling Technology, 13092 S, 1:500); anti-MRP2 (Abcam, ab3373, 1:500); anti-BCAT (Santa Cruz, sc-7199, 1:500); anti-A1AT (Bethyl, A80-122A, 1:250); anti-HNF4A (Abcam, ab201460, 1:250); anti-CYP3A4 (Thermo Fisher, MA5-17064, 1:250); anti-ZO1 (Thermo Fisher, PA5-19090, 1:250). Organoids were washed with PBS three times and subsequently incubated with the appropriate secondary Alexa Fluor antibodies diluted in 2.5% BSA-PBS for 3 hours at RT. After one wash with PBS, organoids were incubated with 1 μg/ml DAPI (Thermo Fisher, 62248), and cell membranes were optionally stained with Phalloidin-Atto 647N (Sigma–Aldrich, 65906, 1:1000), both in PBS for 20 min at RT. After two washes with PBS, organoids were transferred to a 96-well Sensoplate and imaged on a Leica Sp8 microscope.

Cells were harvested, washed in cold PBS, and lysed in Western/IP lysis buffer (Beyotime, Shanghai, China). The whole-cell lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. Protein concentrations were measured using the Bradford method. Proteins were separated via SDS-PAGE, and transferred onto a nitrocellulose membrane. The membranes were blocked using 5% evaporated milk and 1% Tween-20 in PBS for 1 h and were incubated with primary antibodies dissolved in PBS containing 1% Tween-20 overnight at 4 °C. The primary antibodies were anti-YAP (Abcam, Hong Kong, China, #ab52771), anti-β-catenin (Abcam, #ab32572), anti-TGF-β (Abcam, #ab31013), anti-STAT3 (Abcam, #ab68153), anti-HNF4a (Abcam, #ab41898), anti-c-Myc (Abcam, #ab32072), anti-FOXO1 (Abcam, #ab39670) and anti-GAPDH (Cell Signaling Technology (CST), Boston, MA, USA, #5174). On the second day, the blots were incubated with the appropriate secondary antibody: IRDye 800CW goat anti-mouse IgG (LICOR, Lincoln, NE, USA, #926-32210), IRDye 800CW goat anti-rabbit IgG (LICOR, #926-32211), anti-rabbit IgG, HRP-linked antibody (CST, #7074) or anti-mouse IgG, HRP-linked antibody (CST, #7076). The signals were detected using the Odyssey two-color infrared laser imaging system (LICOR) or HRP-based chemiluminescence analysis.

Paraffin-embedded tissue sections (3μm) were deparaffinized and rehydrated in graded alcohol concentrations. After antigen retrieval using sodium citrate buffer, sections were incubated with anti-CYP3A4, anti-CYP2D6, anti-GSTA2 and anti-MRP2 (All from proteintech, USA) and anti-NTCP (Aviva Biosystems, USA) antibodies overnight at 4^oC. Stained tissue sections were visualized using HRP Conjugated goat anti-Rabbit IgG (H+L) secondary antibody and 3, 3-diaminobenzidine (DAB) (Both purchased from DAKO, Denmark). For monolayer culture, differentiated and undifferentiated iHepLPCs cells were cultured in 12-well plates and then fixed with 4% Paraformaldehyde. After blocking and permeabilization, cells were incubated with anti-CYP3A4 (proteintech, USA) and anti-HNF4a (Abcam, UK) antibodies overnight at 4^oC, and then visualized using Alexa Fluor 488 Conjugated goat anti-Rabbit IgG (H+L) secondary antibody or Alexa Fluor 555 Conjugated Donkey Anti-Mouse IgG(H+L) secondary antibody and 4',6-diamidino-2-phenylindole (DAPI) (Both purchased from Beyotime Biotechnology, China). PAS staining of the monolayer culture was carried using the Glycogen Stain kit (Nanjing Jiancheng Bioengineering Institute), following the instructions provided by the manufacturer. Images were captured with Nikon Ta2-FL.