As described by others,19 (link) IHC was used to evaluate the Sirt3 protein expression in glioma tissues. Briefly, deparaffinized and rehydrated slides were treated in 10 mM citrate buffer (pH 6.0) at 95°C for antigen recovery, followed by quenching the endogenous peroxidase activity with 3% H2O2 incubation. Five percent FBS was used for blocking the nonspecific binding sites. Slides were then incubated with primary antibody (anti-Sirt3; Abcam, Cambridge, MA, USA; #ab86671; 1:200) at 4°C overnight. DAB staining kit (Tiangen, China) was used to detect the immunoreactivity according to the manufacturer’s instructions.
The level of Sirt3 was determined by the degree of staining intensity and the percentage of positively stained cells. Examination and scoring were performed by two pathologists independently. In brief, weak staining, moderate staining, and strong staining were scored as 1, 2, and 3, respectively. The percentages of positively stained cells were scored as follows: 1 for less than 20%, 2 for 20%–50%, and 3 for 50%–100%. The final IHC score was defined by multiplying the the two scores above, ranging from 1 to 9. Tissues with a final score no more than 4 were regarded as low-expression cases; otherwise, it will be grouped into the Sirt3 high-expression group.
The level of Sirt3 was determined by the degree of staining intensity and the percentage of positively stained cells. Examination and scoring were performed by two pathologists independently. In brief, weak staining, moderate staining, and strong staining were scored as 1, 2, and 3, respectively. The percentages of positively stained cells were scored as follows: 1 for less than 20%, 2 for 20%–50%, and 3 for 50%–100%. The final IHC score was defined by multiplying the the two scores above, ranging from 1 to 9. Tissues with a final score no more than 4 were regarded as low-expression cases; otherwise, it will be grouped into the Sirt3 high-expression group.
