3 3 diaminobenzidine (dab)

To identify HA, specimens were treated with proteinase K (Dako), endogenous peroxidase was blocked, and sections were stained using a biotinylated HA-binding protein (1:50 dilution; Hokudo) at room temperature for 1 h. Sections were then treated with avitin–biotin peroxidase complex solution (Nichirei Biosciences Inc.) and 3,3′-diaminobenzidine (Dako) according to the manufacturer’s instructions.
To detect CS, specimens were treated with 1.25 U/ml of chondroitinase ABC (Sigma-Aldrich), and sections were incubated with primary antibodies against CS-C4 (2B6) and CS-C6 (3B3) (1:20 dilution; Cosmo Bio. Ltd). To detect HAS2 and COX2, sections were incubated with primary antibodies against HAS2 (1:100 dilution; Abgent Biotech) and COX2 (1:100 dilution; Cayman Chemical Co.).
After microwave antigen retrieval and blockade of endogenous peroxidase, immunostaining was performed with the appropriate primary antibody at room temperature for 2 h. Sections were then treated with EnVision (Dako), according to the manufacturer’s instructions, as well as 3,3′-diaminobenzidine. Sections were counterstained with hematoxylin.

Tissues were isolated and fixed in 4% formalin. Tissues were dehydrated, cut into 4 μm sections, and stained with hematoxylin and eosin. For immunohistochemical stainings, fixed sections were rehydrated and incubated with primary antibodies. The following primary antibodies were used: mouse anti-p120 (1:500; #610134; BD Biosciences) and rabbit anti-Lamin A (1:200; #L1293; Sigma). For mouse tissue, endogenous peroxidases were blocked with 3% H₂O₂ and biotin-conjugated secondary antibodies were used, followed by incubation with HRP-conjugated streptavidin–biotin complex (DAKO). Substrate was developed with DAB (DAKO). For human tissue, stainings were performed using a Ventana BenchMark ULTRA (Roche) and detections were performed using DAB (DAKO) and NovaRED (#SK-4805; Vector Labs). All scoring was done blinded to patient characteristics and results of other staining by two independent observers. Expression of p120 was scored as membranous and/or cytosolic (0 (lowest), 1, 2 and 3(highest)). Expression of p120 was considered lost when the membranous score and the cytosolic score ≤1. Imaging was performed using a Nikon Eclipse E800 microscope mounted with a Nikon digital camera DXM1200.

For immunohistochemistry, formalin-fixed tissues were processed and paraffin-embedded, 4-μm thick sections prepared on charged slides and dewaxed by five changes of xylene, hydrated through decreasing grades of alcohols in water. Cleared sections were blocked with 3% hydrogen peroxide to remove endogenous peroxidase. Antigen retrieval was achieved with Tris-EDTA pH of 9.0.
CD71 (transferrin receptor) staining: Primary CD71 antibody (ThermoFisher, cat no. 13–6800 clone H68.4) was added at 1:300 for 1 hour. HRP-Anti-mouse IgG was used as the secondary antibody. Color development was done using DAB (DAKO, cat no. K3468)
CD3 and CD8 dual staining were performed as per MACH 2 Doublestain Cocktail kit (Intermedico cat no. BC-MRCT525G). A cocktail of both primary antibodies, CD8a (Biolegend, 1 in 100 cat no. 201701, Clone OX-8) and CD3 (1 in 300 DAKO, cat no. A0452) were incubated for 1-hour, and the MACH 2 Doublestain Cocktail (Intermedico cat no. BC-MRCT525G) added as per kit instructions. Colors development was achieved using DAB (DAKO cat no. K3468) for CD3and Warp Red (Intermedico cat no. BC-WR806H) for CD8, counterstaining with Mayer’s Hematoxylin. Sections were mounted with MM 24 Leica mounting medium (cat no. 3801120). CD3 and CD8 double-stained cells are CD8+ T-cells. Quantification was by the automated HALO software (Indica Labs).

For CD3 quantification, liver samples containing tumor nodules were embedded in Tissue-Tek OCT (Sakura, Zoeterwoude, The Netherlands). Samples were air dried, and fixed in 4% formaldehyde for 5 min. Endogenous peroxidase was quenched with 3% H₂O₂ in methanol for 10 min. Tissue sections were incubated o/n at 4°C with anti-human CD3 (clone SP7, Lab Vision Corporation, Fremont, CA) diluted 1∶100 in TBS. Then, sections were incubated with anti-rabbit EnVision HRP-polymer (Dako, Carpinteria, CA) and revealed using DAB+ (Dako). Sections were lightly counterstained with Harris hematoxilin. For pAKT evaluation, formalin-fixed paraffin-embedded liver sections (3 µm thick) were dewaxed and hydrated. Antigen retrieval was performed for 30 min at 95°C in 0,01 M Tris-1 mM EDTA (pH = 9) in a Pascal pressure chamber (S2800, Dako). Endogenous peroxidase was blocked with 3% H₂O₂ in deionized water. Sections were incubated overnight at 4°C with anti-phosphoAKT (1∶50, Cell Signaling, 4060). After rinsing in TBS-T, the sections were incubated with goat anti-rabbit EnVision (Dako; Glostrup, Denmark) for 30 min and peroxidase activity was revealed using DAB+ (Dako). Finally, sections were lightly counterstained with Harris hematoxylin, dehydrated, and coverslipped.

The immunoperoxidase method for CD68, CD83, and DC-sign was performed as described for single immunohistochemistry. Tissue sections were then re-treated with Tris-EDTA antigen retrieval solution at 95 °C for 20 min to inactivate antibodies and enzymes used in the first step. Then, immunohistochemistry for LAP was performed. The combination of chromogens used was as follows: DAB (brown; DAKO), Vector SG (dark blue/gray; Vector Laboratories, Burlingame, CA) and Vulcan Fast Red (red; Biocare, Concord, CA), DAB (brown; DAKO). For Vulcan Fast Red, we used anti-mouse simple stain conjugated with alkaline phosphatase (Nichirei).