Anti afp

Cells were fixed with 4% paraformaldehyde diluted in PBS for 20 min and permeabilized with 0.1% Triton-X 100 diluted in PBS for 10 min at room temperature. Then cells were blocked in 10% goat serum with 1% bovine serum albumin (BSA) for 60 min on the shaker, followed by anti-HBc (Austral, United States), anti-HA (Sigma, United States), anti-nanog (Santa Cruz, Dallas, TX, United States), anti-FoxA2 (Sigma-Aldrich, St. Louis, MO, United States), anti-HNF4a (Cell Signaling), anti-AFP (Sigma-Aldrich, St. Louis, MO, United States) or anti-ALB (Cedarlane, Burlington, Canada) antibodies in 1:500 dilution with 1% BSA in PBS incubated at 4°C over night. The next day, the primary antibody was washed away and the 594 or 488 fluorescent anti-rabbit secondary antibody and 5 mg/ml 4′,6-DAPI with 1:20,000 dilution were added and incubated at 37°C for 30 min. Then, after washing the secondary antibody away, the cells were observed by fluorescent microscopy (Wu X. et al., 2012 (link)).

The following antibodies were used for immunofluorescence staining or western blot analyses: anti-FoxA2 (used at 1:400, Cell Signaling), anti-HNF4α (used at 1:500, Cell Signaling), anti-AFP (used at 1:1000, Sigma-Aldrich), anti-ALB (used at 1:1000, Cedarlane, Burlington, Canada), anti-ZO-1 (used at 1:1000, Thermo Fisher), anti-E cadherin (used at 1:500, Cell Signaling), anti-SR-BI (used at 1:100, Novus Biologicals), anti-BCRP (used at 1:100, Millipore), anti-MRP2 EAG5^{48 (link)} (used at 1:200, a kind gift from Anne Nies, IKP Stuttgart^{48 (link)}), anti-Apo-CIII (used at 1:500, Abcam), anti-CYP8B1 (used at 1:100, Abcam), anti-ORF2 (used at 1:400, a kind gift from Suzanne U. Emerson, NIH) and anti-HAV capsid (used at 1:1000, a kind gift from Stanley M. Lemon, UNC School of Medicine). Alexa Fluor 488 and 549 anti-mouse (used at 1:1000) and Alexa Fluor 488 and 549 anti-rabbit (used at 1:1000) antibodies were purchased from ThermoFisher. Alexa 594-conjugated transferrin was purchased from ThermoFisher. Tenofovir, Tenofovir disoproxil fumarate, and Emtricitabine were obtained through the AIDS Reagent Program, Division of AIDS, NIAID, NIH. Elvitegravir and Cobicistat were purchased from SelleckChem. Sofosbuvir was purchased from Acme Bioscience. BX795, oleic acid and lomitapide were purchased from Sigma-Aldrich.

Huh-7 cell line (AFP positive) was a gift from the Department of Cell Biology, Peking University Health Science Center. Monoclonal antibody against AFP (anti-AFP) was purchased from Sigma (St. Louis, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Invitrogen (California, USA). Monoclonal antibodies against VEGF, VEGFR-2 (KDR), MMP-2, and MMP-9, as well as anti-β-actin were purchased from Abcam (Cambridge, MA, USA).