IHC Staining Quantification and Analysis

IHC was conducted as previously described (24 (link)). Tissue sections (thickness, 4 μm) were deparaffinized in xylene and rehydrated through graded alcohol. Endogenous peroxidase activity was blocked by incubation in 3% H₂O₂ for 10 min at room temperature. The specific antibodies (Abcam) were used as the primary antibodies by using a streptavidin peroxidase-conjugated method. And staining scores were observed in terms of staining intensity and percentage of positively stained cells. Staining intensity was divided into 4 grades: 0 (no staining), 1 (weak staining), 2 (intermediate staining), or 3 (strong staining). The staining percentage were divided into the following grades: 0 (<5% positive), 1 (<25% positive), 2 (25–50% positive), 3 for (51–75% positive), and 4 (>75% positive) (25 (link)). Ten independent fields (×400) were randomly selected to obtain an average score.