Ab108422

For histopathological examination, the liver tissue sections were stained with haematoxylin-eosin (H&E). Sirius red was used to stain for collagen. Immunohistochemistry was performed using 4–5 mm paraffin-embedded thick liver sections, and observed under a photomicroscope. Immunofluorescence staining were observed under a laser scanning confocal fluorescence microscope (Carl Zeiss, Jena, Germany). Antibodies were used as follow: FOXA2 (ab108422, Abcam), α-SMA (BM0002, Boster), α-SMA (MO85129, Dako), CHOP (sc-575, Santa Cruz) and Cleaved Caspase 3 (Asp175,9664, Cell Signaling). Areas of positive stained sites were measured by using image analyses software Image-Pro Plus 6.0 (Media Cybernetics).

Cells were lysed in RIPA buffer (1% w/v Triton X-100, 0.1% w/v SDS, 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% sodium deoxycholate) supplemented with protease and phosphatase inhibitors (Roche, Switzerland). Equal amounts of protein (40 μg/lane) were loaded and resolved via 12% SDS-PAGE (50 V, 3 h) and then transferred to a nitrocellulose membrane (Perkin Elmer, USA). Membranes were blocked with 5% non-fat milk in Tris-buffered saline for 1 h at room temperature and incubated overnight serially at 4°C with the following primary antibodies: GAPDH (loading control; ab8245; 1:500; Abcam), HNF3β (ab108422; 1:1,000; Abcam), IL6 (ab6672; 1:500; Abcam), JAK1 (ab47435; 1:500; Abcam), and STAT3 (ab68153; 1:1,000; Abcam). The membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary goat-anti-rabbit (1:5,000; sc-2004, Sigma, USA) and goat-anti-mouse (sc-2005) antibodies (1:5,000; Sigma, USA) at room temperature for 1 h. Finally, the blots were visualized with enhanced chemiluminescence (Amersham Biosciences, USA) in according with the manufacturer's protocol and detected using an Image Quant LAS 4000 Mini Bimolecular Imager (GE Healthcare, USA). Each experiment was performed in triplicate.

Immunohistochemistry was performed on tissue microarray (TMA) sections. TMA slides were stained for FOXA1 (Abcam ab170933, 1:100 dilution with 10 mM NaCitrate antigen retrieval) and FOXA2 (Abcam ab108422, 1:500 dilution with 10 mM NaCitrate antigen retrieval) using a standard procedure^{67 (link)}. Rabbit IgG was used as a negative control. Nuclear staining intensity was assigned levels 0, 1+, 2+, or 3+ and H-scores were calculated as: [1 x (% of 1+ cells) + 2 x (% of 2+ cells) + 3 x (% of 3+ cells)]. Evaluations were performed in a blinded fashion.

At each differentiation stage, cells were fixed with 4% PFA for 15 min at room temperature then washed three times with PBS. The slides of cells were permeabilized with PBS + 0.5% Triton X-100 for 20 min and washed three times with PBS. Slides were blocked with 10% goat serum for 30 min and incubated with the primary antibodies at 4°C overnight (>16 h) diluted in PBS. Following incubation, the slides were rinsed with PBS and incubated with secondary antibodies at 37°C for 1 h. The slides were rinsed with PBS and the cell nucleus was stained with DAPI (Sigma Aldrich). The images were visualized using an Olympus BX41 fluorescence microscope. Dilutions and Catalog numbers for primary antibodies were as follows: Anti-Oct4 antibody (1:200 dilution) (Abcam, ab18976), Anti-Nanog antibody (1:200 dilution) (Abcam, ab80892), Anti-FOXA2 antibody (1:300 dilution) (Abcam, ab108422), Anti-SOX17 antibody (1:50 dilution) (Abcam, ab191699), Human/Mouse/Rat SOX2 antibody (1:200 dilution) (R&D Systems, AF2018), Anti-TTF1 antibody (1:200 dilution) (Abcam, ab76013), Anti-SOX9 antibody (1:200 dilution) (Abcam, ab26414). The secondary antibody: Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (1:500 dilution) (Abcam, ab150077), Rhodamine (TRITC)-conjugated AffiniPure Bovine Anti-Goat IgG (H+L) (1:100 dilution) (Jackson ImmunoResearch, 805-025-180).