Abc peroxidase and diaminobenzidine

The tumor tissue was taken out, fixed with paraffin, embedded, and sectioned, the section was deparaffinized in xylene, and rehydrated with a graded concentration of ethanol and distilled water. Then use 0.3% hydrogen peroxide (China) to quench the endogenous peroxidase activity, and heat the strong antigen recovery solution to 37 °C to recover the antigen. A total of 5% goat serum (Solarbio, China) was used to block non-specific proteins. The first antibody was incubated overnight at 4 °C, and the second antibody was incubated at room temperature for 60 min. Then, the slides were incubated with ABC peroxidase and diaminobenzidine (ZSGBBio). Next, the slides were counterstained with Mayer hematoxylin solution (Solarbio) for nuclear staining.

The antibiotin protein‐biotin method got accustomed to perform immunostaining on paraffin‐embedded sections. Slides were dewaxed in xylene, then rehydrated in graded ethanol, then the endogenous peroxidase activity was then quenched with 0.3% hydrogen peroxide (China), and the strong antigen recovery solution was heated to 37°C to recover the antigen. A total of 5% goat serum (Solarbio, China) was used to block nonspecific proteins. Primary antibodies (1:100 dilutions) were used to incubate sections at 4°C overnight, subsequently the appropriate biotinylated secondary antibody was added (1:100 dilutions) (ZSGBBio, China) at 37°Cfor 60 min. Then, slides were then hatched with ABCperoxidase and diaminobenzidine (ZSGBBio). Next, the slides were counter‐stained for nuclear staining by Mayer hematoxylin solution (Solarbio). For going on H&E staining, the slides were deparaffinized and rehydrated. Next, the slides were stained by nuclear staining, subsequently restaining using HE kit (Solarbio). The images were taken with an inverted microscope (Olympus, Japan). The human tissue samples used in this study research have complied with the relevant national and institutional policies.

The antibiotin protein-biotin method got accustomed to performing immunostaining on paraffin-embedded sections. Slides were dewaxed in xylene, then rehydrated in graded ethanol, then the endogenous peroxidase activity was then quenched with 0.3% hydrogen peroxide (China), and the strong antigen recovery solution was heated to 37°C to recover the antigen. 5% goat serum (Solarbio, China) was used to block nonspecific proteins. Primary antibodies (1:100 dilutions) were used to incubate sections at 4°C overnight, subsequently the appropriate biotinylated secondary antibody was added (1:100 dilutions) (ZSGBBio, China) at 37 °C for 60 minutes. The, slides were then hatched with ABC peroxidase and diaminobenzidine (ZSGBBio, China). Next, the slides were counter-stained for nuclear staining by Mayer hematoxylin solution (Solarbio, China). For going on H&E staining, the slides were deparaffinized and rehydrated. Next, the slides were stained by nuclear staining, subsequently re-staining using HE kit (Solarbio, China). The images were taken with an inverted microscope (Olympus, Japan). The human tissue samples used in this study research has complied with the relevant national and institutional policies.