Ab10241

The imHCs were cultured onto 96-well CellCarrier-96 optic black plates (PerkinElmer, Waltham, MA, USA) and stained with antibodies against hepatocyte markers: Albumin (ALB) (1:100 dilution, ab10241, Abcam), α-fetoprotein (AFP) (1:100 dilution, SC8399, Santa Cruz Biotech, Dallas, TX, USA), LDLR (1:100 dilution, SC373830, Santa Cruz Biotechnology), sodium taurocholate cotransporting polypeptide (NTCP) (1:100 dilution, ab131084, Abcam), MRP2 (1:100, AB3373, Abcam) and hepatocyte nuclear factor-4α (HNF-4α) (1:100 dilution, SC6556, Santa Cruz Biotech). For detecting HBV infectivity, infected hepatocytes were stained with antibodies against HBV proteins: HBcAg (1:100 dilution, ab8637, Abcam), HBsAg (1:100 dilution, ab20758, Abcam). Hepatocytes were then incubated with goat anti-mouse Alexa Fluor^® 488-conjugated (1:500 dilution, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), goat anti-rabbit Alexa Fluor^® 488-conjugated (1:500 dilution, Invitrogen), or donkey anti-goat Cy3-conjugated secondary antibody (1:500 dilution, BioLegend, San Diego, CA, USA). Hepatocyte nuclei were stained with 2 µM Hoechst 33342 (Thermo Fisher Scientific, MA). Mouse IgG2a, mouse IgG1, rabbit IgG and goat IgG were used as negative control for staining. Fluorescence images were captured by an Operetta High-Content Imaging System (PerkinElmer, MA) with a 40× objective lens.

Protein samples were prepared from human serum (DS and age-matched controls, n = 20). Twenty μg protein samples were separated on 4–12% Nu-PAGE Bis-tris (Bis (2-hydroxyethyl)-amino-tris (hydroxymethyl)-methane) gradient gels and transferred to 0.2 μm pore size PVDF for Aβ42, hepcidin and S100β, and 0.45 μm pore size PVDF membranes for SOD1, FTL, FPN, TREM2 and albumin using the NuPAGE electrophoresis system (Invitrogen). Membranes were incubated with different antibodies, i.e., anti-β amyloid (Covance, SIG 39320), SOD1 (Abcam, Cambridge, UK, ab252426), S100β (Abcam, ab218514), anti-ferritin light chain (FTL, Abcam, ab69090), anti-FPN (Abcam, ab85370), anti-hepcidin 25 (Abcam, ab30760), anti-TREM2 (Abcam, ab86491) and anti-albumin (Abcam, ab10241) antibody, in blocking buffer for 24 h at 4 °C, and then washed three times with 0.1 M Tris saline buffer containing 1% Tween 20 (TBST), followed by incubation for 1 h at RT with HRP-conjugated secondary antibodies, anti-rabbit IgG (1:3000, DAKO) or anti-mouse IgG (1:3000; DAKO) antibodies. Binding was detected with ECL Plus chemiluminescence reagents.

In order to observe specific markers and morphological changes of the embedded cells in scaffolds, immunofluorescence staining was performed. At given time points, cell-laden scaffolds were collected, fixed with 4% formaldehyde, washed in HBSS, permeabilized with 1% Triton X100 and then blocked with 3% bovine serum albumin (BSA). For specific biomarker staining, different antibodies were applied: mouse Anti-Human Serum Albumin antibody (ab10241; Abcam, Germany, dilution 1:2000) labeled with AlexaFluor 568-tagged goat anti-mouse secondary antibody (ab175473; Abcam), mouse Anti-Human Cytokeratin 19 monoclonal antibody (ThermoFisher scientific, Germany, dilution 1:500) detected via AlexaFluor 568-tagged goat anti-mouse secondary antibody, rabbit Anti-Human α1 antitrypsin polyclonal antibody (ThermoFisher scientific, Germany, dilution 1:250) detected via AlexaFlour 488 goat anti-rabbit secondary antibody (ThermoFisher scientific). Cell nuclei were stained with DAPI and cytoskeletons with Phalloidin-iFluor 488 Reagent. Expression of cell surface markers and morphology was observed with a Leica TCS SP5 cLSM.