Envision detection system

Immunohistochemistry was performed following a standard protocol. Briefly, slides were deparaffinized with xylene and ethanol, and the endogenous peroxidase was blocked by 3% H₂O₂ for 10 minutes. After being incubated in retrieval buffer and boiled for 3.5 minutes, slides were washed with PBS for 3 times and blocked with 5% normal serum. Then the slides were incubated with primary antibody at 4 °C overnight followed by 60 minutes-treatment of MaxVision HRP solution (MXB Biotechnology, cat# 5020). After being stained with DAB Peroxidase Substrate (MXB Biotechnology, cat# 0031), the antigen levels were detected using EnVision Detection System (Agilent Technologies, K5007).

In situ hybridization assays were performed to evaluate the STEAP3-AS1 levels in the CRC xenograft model. Sections were deparaffinized with xylene and ethanol, and the endogenous peroxidase was blocked by 3% H₂O₂ for 10 minutes at room temperature. After being incubated with 3% citric acid and freshly diluted pepsin for about 60 s at 37 °C, slides were washed 3 times with PBS and fixed with 1% formaldehyde with addition of 0.1% DEPC for 10 min at room temperature. The sections were then pre-hybridized at 40 °C for 2 hours in a hybrid box with 20 mL 20% glycerinum placed in the bottom. Twenty microlitre hybridization liquid was then added and left at 40 °C overnight. After being washed successively with 2 × SSC, 0.5 × SSC, 0.2 × SSC (15 minutes for each), the sections were blocked with blocking reagent for 30 min at 37 °C. Next, sections were incubated with biotin-digoxigenin for 1 hour at 37 °C followed by Strept Avidin-Biotin Complex (SABC) for 20 min at 37 °C. After being incubated with biotin peroxidase, sections were subjected to DAB. This was followed by hematoxylin redye, dehydration using graded ethanol and vitrification with dimethylbenzene. Sections were analysed using an EnVision Detection System (Agilent Technologies, K5007).

Briefly, the human HCC tissue microarray (TMA) was deparaffinized with xylene and ethanol and further blocked by 3% H₂O₂ for 10 min at room temperature. Then, the TMA was incubated in retrieval buffer and boiled for 3.5 min. After three washes with PBS, the TMA was blocked with 5% normal serum and incubated with primary antibody at 4°C overnight. Next, TMA was treated with MaxVision HRP solution (MXB Biotechnology, cat# 5020) for 60 min, followed by staining with DAB Peroxidase Substrate (MXB Biotechnology, cat# 0031). The EnVision Detection System (Agilent Technologies, K5007) was used to detect antigen expression.

Immunohistochemical staining (IHC) was performed on 4-μm FFPE tissue sections using Envision Detection System (DAKO/Agilent, Glostrup,), according to manufacturer’s protocol. Tissue sections were deparaffinized with xylene and rehydrated in a series of decreasing concentration ethanol solutions. Heat-induced epitope retrieval was performed in a 96 °C water bath, for 30 min in Target Retrieval Solution pH 9 (DAKO). Tissue samples were incubated with the primary antibodies against CD3 (clone F7.2.38; dilution 1:50; DAKO/Agilent) CD8 (clone D8A8Y; dilution 1:200; Cell Signaling Technology) and CD4 (clone; dilution 1:500; Cell Signaling Technology) for 1 h in RT. Color reaction product was developed using 3,3′-diaminobenzidine tetrahydrochloride as a substrate and nuclear counterstaining was obtained with hematoxylin.