Ab66596

Liver tissues were fixed in 10% neutral buffered formalin, paraffin embedded, and cut into 4 µm sections. Glypican-3 (GPC3) expression was analyzed using a rabbit polyclonal antiserum raised against GPC3 (ab66596) (Abcam, Cambridge, UK). Slide images were captured using a Nanozoomer slide scanner (Hamamatsu) and analyzed using Image J software.

After deparaffinization and rehydration, antigen retrieval was applied by microwaving for 10 min with 0.01 M citric acid buffer. The endogenous peroxidase was destroyed by hydrogen peroxide. Primary antibodies were reacted at 4 °C overnight. The secondary antibody (EnVision System-HRP; Dako, Glostrup, Denmark) was incubated for 1 h at room temperature. Subsequently, staining with 3,3′-diaminobenzidine (Dako) for 3 min was performed. For immunohistochemistry of tumor lesions, rabbit polyclonal antibodies for GS (11037–2-AP; Proteintech, Chicago, IL, USA; 1:200 dilution), GPC3 (ab66596; Abcam, Cambridge, UK; 1:200 dilution), HSP70 (10995–1-AP; Proteintech; 1:200 dilution), and ARG1 (AV45673; Sigma-Aldrich, Steinheim, Germany; 1:200 dilution) were used as primary antibodies.

Paraffin-embedded liver sections were deparaffinized and rehydrated with xylene and graded concentrations of ethanol. After microwave antigen retrieval and endogenous peroxidase activity blocking, primary antibodies against alpha fetoprotein (AFP, 14550-1-AP; Proteintech, Rosemont, IL, USA), CD34 (ab81289), cytokeratin 19 (CK19, ab52625), and glypican 3 (GPC3, ab66596) (Abcam, Cambridge, UK), and the secondary anti-rabbit antibody (PV-6002; Zhongshan Golden Bridge Biotechnology, Beijing, China) were used to incubate the tissue sections. Immunostaining was detected using 3,3′-diaminobenzidine staining and counterstaining with 10% Mayer hematoxylin.

For confirmation of HBV pre-S/S protein expression, monoclonal anti-HBs antibody (M-21853, Genzyme Diagnostics, San Carlos, CA, USA), goat polyclonal anti-HBs antibody (bs-1557G, Bioss, Woburn, MA, USA), monoclonal anti-pre-S2 antibody (sc-23944, Santa Cruz Biotechnology, Dallas, TX, USA) were used in western blot or IHC analysis. For characterization of HCC and oncogenic molecular pathway, anti-glypican-3 antibody (ab66596, Abcam, Cambridge, MA, USA), anti-GRP78 antibody (ab108615, Abcam, Cambridge), anti-ERK1/2 antibody (137F5, Cell Signaling Technology), anti-phosphorylated extracellular signal-regulated kinases 1/2 antibody (20G11, Cell Signaling Technology), β-actin antibody (GeneTex, Irvine, CA, USA), anti-B-cell lymphoma-extra large antibody (54H6, Cell Signaling Technology), anti-cyclin E antibody (sc-481, Santa Cruz Biotechnology), anti-CSMD3 antibody (sc-68281, Santa Cruz Biotechnology) and anti-GAPDH antibody (GeneTex) were used. Cell proliferation was also assessed using anti-Ki-67 antibody (Merck Millipore, Temecula, CA, USA). Apoptotic cells in liver tissue sections were detected using the DeadEnd Fluorometric TUNEL System (Promega Corporation, Madison, MI, USA) according to the manufacturer's manual. Cell apoptosis was also assessed using anti-activated caspase 3 antibody (EnoGene Biotech, New York, NY, USA).

Liver tissues were fixed in 10% neutral buffered formalin, paraffin embedded and cut into 4 μm sections. Sections were H&E stained and assessed for tumour grade by pathologist Professor Robert Goldin (Imperial College London). Necrosis was determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) staining (ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit, Milipore). Hepatocyte proliferation was determined by staining with polyclonal rabbit anti-MCM4 antibody at a 1/100 dilution (Abcam, ab84153). PD-1 expressing lymphocytes were identified using a rabbit polyclonal anti PD-1 antibody at 2.5 μg ml⁻¹ (Biorad ahp1706). RAE-1 expression was analysed using a polyclonal goat anti-mouse RAE-1 antibody at 10 μg ml⁻¹ (R&D Systems). HCC markers were stained using anti-Hsp70 at 1 μg ml⁻¹ (EPR16892) and rabbit polyclonals raised against Glypican-3 at 5 μg ml⁻¹ (ab66596) and Anti-Glutamine Synthetase at 1 μg ml⁻¹ (ab73593) from abcam. Slide images were captured using a Nanozoomer slide scanner (Hamamatsu) and analysed using Image J software.