Catalyzed sign amplification system kit

The tissue samples were placed into citrate buffer and heated in a microwave oven. Slides were then incubated with anti-VEGFA (1:100, Abcam, Cambridge, England), anti-MELK (1:500, Abcam, Cambridge, England), or anti-CD33(1:100, Abcam, Cambridge, England) antibody at room temperature for 1 h. Following washing, each section was briefly counterstained using a catalyzed sign amplification system kit (Dako code k5007). The images were captured under the same conditions.

After the dewaxed paraffin‐embedded tissue sections were subjected to rehydration, the samples were placed into citrate buffer (10 mm, pH 6.0) and heated twice in a microwave oven. Sections were incubated for 10 min with 3% H₂O₂ and washed in PBS. After being blocked by 10% normal goat serum for 0.5 h, samples were incubated with the purified anti‐Ki67, anti‐PCNA, anti‐E‐cadherin, anti‐N‐cadherin and normal control IgG at 4 °C for 12 h. Following washing, each section was stained using a catalyzed sign amplification system kit (Dako code k5007) and observed by looking at photos of the sections.

Paraffin tissue sections were dewaxed, rehydrated, and placed in 10 mmol/L of citrate buffer (pH 6.0), then heated twice in a microwave oven for 5 min each. Sections were incubated with 3% H₂O₂ for 10 min, washed with PBS, blocked with 10% normal goat serum for 30 min, and then incubated with 4 mg/L purified anti-SLUG, anti-SNAIL, anti-β-catenin, and normal rabbit IgG as a control at 4°C overnight. After washing, the sections were stained with the catalyzed sign amplification system kit (DAKO code k5007) and visualized through photos taken.