Hep par 1

Immunohistochemical staining was performed on 5-micron cut sections of formalin-fixed, paraffin-embedded (FFPE) tissue blocks of all cases on Ventana staining system (Ventana Medical Systems, Tucson AZ, USA). All cases were stained with INI1 (monoclonal mouse antibody; 1:400; BD Bioscience, San Jose, CA, USA), Hep-par1 (mouse monoclonal antibody; 1:200; Dako, Santa Clara, CA, USA), Arginase (rabbit monoclonal antibody; 1:100; Sigma-Aldrich, St. Louis, MO, USA) and glypican-3 (mouse monoclonal antibody; ready to use; Sigma-Aldrich, St. Louis, MO, USA) antibodies.

Representative 4-μm serial sections were prepared from 10% formalin-fixed, paraffin-embedded tissue blocks. Deparaffinization was conducted by treating the cells with xylene for 10 minutes; 100% ethanol for 1 minutes, 95% ethanol for 1 minutes, 85% ethanol for 1 minutes, and 75% ethanol for 1 minutes. Briefly, all slides were exposed to 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. Microwave antigen retrieval was performed in citrate buffer (pH 6.0) for 5 minutes to increase the immunoreactivity. Microwaving at medium/high heat for 15 minutes, keeping the solution boiling, followed by treatment with 5% skimmed milk in phosphate-buffered saline–0.1% bovine serum albumin for a minimum of 1 hour at room temperature to block nonspecific staining.
Hep Par 1 (cytoplasm of hepatocytes, 1:50, Dako) and CK-19 (cytoplasm of cholangiocytes, 1:50, Dako) were added to HCC and ICC tissue, respectively. Sections were incubated with primary antibodies in a humid chamber at 4 °C overnight, followed by incubation with antimouse peroxidase-conjugated envision antibody at 37 °C for 30 minutes. Immunoreactions were visualized by 3,3-diaminobenzidine as chromogen for 5 minutes at room temperature, followed by light counterstaining with hematoxylin. Tissue structures were visualized by counterstaining with hematoxylin (Bioengineering, Shanghai, Ltd., China).

Processed blood samples were permeabilized with 0.1% Triton-X and blocked with 10% donkey serum for 30 minutes. Immunofluorescence (IF) staining was conducted using primary antibodies against a combination of markers including the following: Glypican 3 (Biocare Medical, California), Glutamine Synthetase (Biocare Medical, California), HepPar-1 (Dako, Denmark), CD44 (Atlas, Sweden), and CD45 (BD, New Jersey). Secondary antibodies conjugated with Alexa Fluor 488, 546, or 647 (Invitrogen) were diluted in 1% BSA and used for detection. The cytoslides were mounted with Prolong Gold Antifade Mountant with 4′, 6-diamidino-2-phenylindole (DAPI) (Thermo Scientific, Massachusetts) to counterstain the cell nuclei and stored at 4 °C until imaging. Slides were observed under 20x magnification using the inverted immunofluorescence microscope (Ti Eclipse, Nikon, New York) with an automated motor stage. Images were reviewed and counted manually.

Tumor cell agarose pellets were prepared from tumor cell lines, fixated in 10% neutral buffered formalin, paraffin embedded, sliced and confirmed as HCC by immunostaining for CK7 (dilution 1:200; Abcam) and HepPar1 (dilution 1:50; Dako)^{24 ,25 (link)}.
The tumor specimens were formalin-fixed and paraffin-embedded according to standard protocol. The endogenous HCCs were stained with H&E for classification as HCC lesions by a board-certified veterinary pathologist (KS). The subcutaneous tumors were stained with H&E and anti-CD31 rabbit antibody (dilution 1:50; Abcam, Cambridge, UK) using an automated BondRxm staining unit. To analyze CD31 staining, 6 representative visual fields (20 × magnification) were allocated over the whole slide of each tumor, and vessel ingates were enumerated and then averaged.

Livers or tumors were harvested, fixed in 10% (v/v) formalin overnight, and embedded in paraffin. Paraffin sections (4 µm) were prepared for staining. The following antibodies were used: AE1 (1:200, Signet Laboratories, 462-01), Hep Par1 (1:100, Dako, M7158), PCNA (1:1000, Abcam, ab18197), Cyclin D1 (1:200, Cell Signaling Technology, 2978), phospho-Stat1 (Tyr701) (1:500, Cell Signaling Technology, 9167), CD45 (1:200, Cell Signaling Technology, 70257), CD3 (1:200, Dako, A0452), F4/80 (1:200, Cell Signaling Technology, 70076), CXCL9 (1:500, Invitrogen, PA5-79115), S100A9 (1:300, Cell Signaling Technology, 73425), and PD-L1 (1:300, Cell Signaling Technology, 13684).