AQP3/CD133/CD44/CD90/EPCAM expression was analyzed in paraffin-embedded specimens obtained from 120 patients. Four-µm-thick tissue sections were deparaffinized in xylene and dehydrated before antigen retrieval for 5 min using an autoclave. The endogenous peroxidase activity was blocked using hydrogen peroxide (0.3%), and non-specific immunoglobulin binding sites were blocked by normal goat serum for 30 min at 37 °C. Tissue sections were incubated with anti-AQP3 (1:1000, Abcam), anti-CD133 (1:200, Abcam), anti-CD44 (1:1000, Abcam), anti-EPCAM (1:200, Abcam), and anti-CD90 (1:200, Abcam) overnight at 4 °C. Then, the sections were incubated with biotinylated goat anti-rabbit IgG as a secondary antibody (Maixin Kit, China) for 1 h at room temperature, followed by incubation with streptavidin–biotin horseradish peroxidase-conjugated (Maixin Kit) for 30 min at room temperature. The peroxidase reaction was developed with 3′-diaminobenzidine tetrahydrochloride (Maixin Kit). The expression levels of the proteins were scored semi-quantitatively according to the percentage of positively stained cells combined with the staining intensity according to our previous study18 (link). The specimens were assessed three times.
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