Tumor-bearing brain tissue were collected from mice of each group. Tissues were fixed in 10% buffered formalin for 24 h following standard procedure for processing, paraffin-embedding, and sectioning to assess PCNA (1:200, Servicebio, GB11010), E-cadherin (1:200, Servicebio, GB14076) by IHC assay. The reaction was visualized using the Servicebio image analysis system, the staining was scored by two independent and experienced pathologists and calculated as the product of the staining intensity. First divides the positive grade: negative without staining, score 0, weak positive light yellow, score 1, medium positive brown, score 2, strong positive brown, score 3 points. Then analyze and calculate the area of weak, medium, and strong positive in the measurement area, the tissue area of the measurement area, the cumulative optical density value of the positive and the positive area. PCNA and E-cadherin quantification for each sample was determined and molecular data using modified H-scores ([{% of weak staining} × 1] + [{% of moderate staining} × 2] + [{% of strong staining} × 3]), to determine the overall percentage of PCNA and E-cad positivity across the entire stained sample, yielding a range from 0 to 300 [22 (link), 23 (link)].
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