2 step detection kit

Human HCC tissue microarrays (TMA) slides consisted of 156 pairs of primary tumor tissues and ANLT specimens. The immunohistochemical-staining assay for LLGL2(1:50), Vimentin(1:100), E-cadherin(1:100) was conducted using a 2-step detection kit (Zhongshan Golden Bridge Biotechnology, Beijing, China).Antibody Vimentin and E-cadherin purchased from HuaAn Biotechnology (Huaan, Hangzhou, China). We evaluated LLGL2 expression with an inverted microscope (Olympus, Japan). The levels of LLGL2 expression were scored on the basis of the percentage of the positively stained tumor cells. The grading score were; 4 indicates >80% positive, 3 indicates 51-80% positive, 2 indicates 31-50% positive, 1 indicates 5-30% positive, and 0 indicates ≤5% positive. The level of LLGL2 protein expression was classified into a high expression group (2 - 4) and a low expression group (0 - 1) for downstream analyses (33 (link)).

Immunohistochemistry was performed with a 2-step detection kit (Zhongshan Golden Bridge Biotechnology, Beijing, China). Deparaffinized sections were subjected to antigen retrieval by 0.125% trypsin and 20 μg/ml proteinase K solution. The sections were then blocked with 5% BSA and treated with the following antibodies: runt-related transcription factor 2 (Runx2; 1:300; orb10256, Biorbyt), rat zinc finger transcription factor Osterix (Osx; 1:300; PA5-40509, Thermofisher scientific), and transforming growth factor- Beta 1 (TGF-β; 1:200; LS-C10852, LSBio). After washing with PBS, the sections were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies, using diaminobenzidine (Zhongshan Golden Bridge Biotechnology, Beijing, China) as chromogen.