3 3 diaminobenzidine tetrahydrochloride dab

Deparaffinized samples were hydrated, followed by 3% hydrogen peroxide blocking for 15 minutes. The specimens were then immersed in 0.01 M boiling citrate buffer and microwaved for one minute in order to extract antigens. Immediately following blocking with 5% BSA (Boster, Wuhan, China) for 40 minutes, the sections were incubated overnight at 4°C with primary antibodies against AR (1:100), β-catenin (1:100) and HMGB-1 (1:100), respectively. Samples were deparaffinized, hydrated, and blocked with 3% hydrogen peroxide for 15 minutes. Then slices were subjected to antigen retrieval by immersing in 0.01 M boiling citrate buffer and heating in a microwave oven for 1 minute. Sections were blocked by 5% BSA (Boster, Wuhan, China) for 40 minutes at room temperature, and further incubated with primary antibodies against AR (1:100), β-catenin (1:100), and HMGB-1 (1:100) at 4°C overnight, respectively.28 (link) Finally, expression of immunoreactivity was visualized by 3,3’-diaminobenzidine tetrahydrochloride (DAB; Boster, Wuhan, China) after process of incubating with horseradish peroxidase (HRP)-conjugated secondary antibodies (Boster, Wuhan, China) at 37°C for 40 minutes. Using hematoxylin as a nuclear counterstain, the slices were then dehydrated in a graded series of alcohol washes and mounted with coverslips.41 (link)

Primary OS and lung metastatic OS tissues from the same patient were collected from Guangxi Medical University Cancer Hospital. IHC staining was performed to identify the protein expression of RBM15 in OS tissues. Briefly, the OS tissue sections were incubated with 3% H₂O₂ for 15 min and blocked by goat serum for another 15 min at room temperature. Subsequently, knee tissue sections were incubated with the primary antibody of RBM15 (1:200 dilution; Proteintech, China) at 37°C for 90 min in the humidified chamber. After being rinsed three times with PBS, OS tissue sections were incubated with the biotin-labeled goat anti-mouse/rabbit immunoglobulin G (IgG) (ZSGb Bio, China) for 30 min. Then, samples were counterstained with hematoxylin (Solarbio, China) after being stained with 3, 3′-diaminobenzidine tetrahydrochloride (DAB; Boster, China). Finally, the tissue sections sealed with neutral resin were imaged using the upright microscope (Olympus BX53, Tokyo, Japan).