Streptavidin horseradish peroxidase complex

Examination of the expression and distribution of Wnt5a, β-catenin, and ROR2 in NSCLC tissues was performed by the immunohistochemical method. Briefly, 4-μm paraffin-embedded sections were deparaffinized and rehydrated. For the blockage of endogenous peroxidase activity, 3% hydrogen peroxide was used. After antigen retrieval, sections were incubated with the primary antibodies against Wnt5a, β-catenin, and ROR2 (each diluted in 1:50) at 4°C overnight. Biotinylated secondary antibodies were then used to treat the tissues sections, followed by incubation with streptavidin–horseradish peroxidase complex (Santa Cruz Biotechnology Inc., Santa Cruz, California, U.S.A.). Immunoreactivity was visualized with diaminobenzidine (Sigma–Aldrich, St. Louis, MO, U.S.A.). The sections were counterstained with Hematoxylin. For blank controls, the primary antibody was omitted.

Immunohistochemical staining was carried out to assess the protein expression pattern of PRMT9 and HIF-1α in paraffin-embedded human OS tissues. Briefly, the paraffin-embedded tissue blocks from OS patients were cut into 4-μm-thick sections and baked at 65° for 30 min. Then the sections were deparaffinized with xylene, followed by rehydrated in the water, submerged into EDTA antigenic retrieval buffer and microwaving was processed for antigen retrieval. Next, endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 15 min, followed by incubation with 1% bovine serum albumin to block the nonspecific binding. The specimens were incubated overnight at 4°C with a rabbit polyclonal antibody against indicated antibodies. The antibody was replaced with normal goat serum as negative controls. After washing with PBST, the tissue slides were incubated with a biotinylated anti-rabbit secondary antibody (Santa Cruz) at room temperature for 30 min, followed by further incubation with streptavidin-horseradish peroxidase complex (Santa Cruz) at room temperature for 30 min. The slides were counterstained with 10% Mayer's hematoxylin and mounted in Crystal Mount.

The paraffin-embedded sections were dewaxed and rehydrated. 3% hydrogen peroxide was used to block peroxidase activity. Sections were incubated throughout the night with primary antibody (human APOE, PD-1, TIGIT, abcam, UK, mouse C1QC, CCR2, CD206, CD86, abcam, UK) at 4 °C. Next, the biotinylated secondary antibody was adopted to treat tissue sections and then incubated with streptavidin-horseradish peroxidase complex (Santa Cruz Biotechnology Inc., USA).