P0186

Paraffin-embedded sections were heated for antigen retrieval in citrate buffer (0.01 M, pH 6.0), and they were incubated with 5% (v/v) goat serum (Zhongshan Jinqiao Biotec) for 30 min to block non-specific binding sites. The primary antibodies including mouse anti-cytokeratin 7 (1:500 dilution, 66483-1, ProteinTech Group, Inc.), mouse anti-E-cadherin (1:500 dilution, ab40772, Abcam), rabbit anti-human CD10 antibodies (1:250 dilution, 18008-1-AP, ProteinTech Group, Inc.) and rabbit anti-human vimentin (1:500 dilution, ab45939, Abcam) were used to identify stromal and epithelial cells and incubated overnight at 4˚C. The slides were incubated with 10 µg/ml FITC-conjugated goat anti-rabbit secondary antibody (1:1,000 dilution, P0186, Beyotime Institute of Biotech) or 10 µg/ml Cy3-conjugated goat anti-mouse secondary antibody (1:1,000 dilution, P0186, Beyotime Institute of Biotech) for 1 h at 37˚C. Nuclei were counterstained with DAPI. Negative control included sections stained with a nonimmune serum in the absence of the primary antibody. Fluorescent images were captured with an inversed fluorescent microscope (IX-71, Olympus Corp.) at room temperature. All images were evaluated with the same setting for brightness and contrast at original magnifications of x100 and x200.