Envision plus detection system

The HCC tissues were serially sectioned at a thickness of 3–5 mm. Then, the tissues were dewaxed, deparaffinized in xylene, and rehydrated. The samples were then boiled for 15 min in a microwave oven for the purpose of increasing antigen retrieval. Then, 3% hydrogen peroxidase was used to block the endogenous peroxidases for 30 min. The slides were incubated with primary antibody CD8⁺ (PeproTech, 67786-1, 1:8000) overnight. Biotin-free horseradish peroxidase-labeled polymer of the Envision Plus detection system (Dako, Denmark) was used in the process of detection. Double immunofluorescence staining was performed as previously described. Paraffin-embedded sections were incubated with the following primary antibodies: F4/80 (Santa Cruz Biotechnology, SC-365340, 1:500), CD206 (PeproTech, 18704-1-AP, 1:800), and CD86 (CST, 91882S, 1:100) at 4°C overnight. Then, the sections were incubated at 37°C with the matched fluorescently labeled secondary antibodies (1:500; Invitrogen; CA, USA) for 30 min.

Formalin-fixed and paraffin-embedded HCC sections with a thickness of 4 μm were dewaxed in xylene and graded alcohols, hydrated and washed in phosphate buffered saline (PBS). Antigen retrieval was done by heat treatment of the deparaffinized sections in a pressure cooker in EDTA-TRIS (PH9.0) for 2 minutes. After the initial processing steps, sections were incubated overnight with primary antibody at 4 °C. The primary antibodies used in IHC were as follows: anti-RTN3 (Sigma, # HPA015649, 1:300 dilution), anti-SOCS2 (Abcam, ab74533, 1:150 dilution), anti-UPB1 (Abcam, ab157195, 1:600 dilution). A subsequent reaction was performed with biotin-free HRP enzyme-labeled polymer from an EnVision plus detection system (DAKO, K5007, Glostrup, Denmark). Positive reactions were visualized with diaminobenzidine (DAB) solution followed by counterstaining with hematoxylin. Negative controls were performed using non-immune goat serum instead of the primary antibodies. The protein expression was assessed by a certified pathologist who was blind to all clinical and biological variables using a semi-quantitative scoring consisting of an assessment of both staining intensity (scale 0–3: 0, none; 1, mild; 2, moderate; and 3, intense.) and the percentage of positive cells (0–4: 0, <5% cells; 1, 6–25% cells; 2, 25–50% cells; 3, 51–75% cells; and 4, >75% cells), yielding an overall score.