Formalin-fixed, paraffin-embedded tumor and adjacent nontumor (>2 cm away from tumor) tissues were sectioned at 4-μm intervals. Immunohistochemical staining was carried out on both tumor and nontumor tissue slides. The slides were deparaffinized in xylene and rehydrated through graded alcohol. Endogeneous peroxidase was then inactivated with 3% hydrogen peroxide at room temperature for 20 minutes. Then the slides were soaked in 0.1 mol/L citrate buffer (pH 6.0) and placed in an autoclave at 121 °C for 2 minutes for antigen retrieval. After washing with PBS (pH 7.4), the sections were blocked with 1% BSA diluted in PBS at 37 °C for 30 minutes, and then incubated with primary antibodies at 4 °C overnight. Then the HRP-conjugated goat anti-mouse/rabbit antibody and DAB (DAKO, Glostrup, Denmark) were used. Finally, the sections were counterstained by hematoxylin and mounted. The following antibodies were used: HNF-1B (1:500, Sigma-Aldrich, St. Louis, MO), K7 (1:200, DAKO, Glostrup, Denmark), K19 (1:200, DAKO, Glostrup, Denmark), EpCAM (1:200, DAKO, Glostrup, Denmark), OV6 (1:40, DAKO, Glostrup, Denmark) and PCNA (1:4000, Cell Signaling Technology, MA, USA).
Double-fluorescence immunostaining of the tumor and nontumor tissue was performed with a sequential fluorescent method as described17 (link). Immunofluorescence was observed with the Olympus IX-71.
Double-fluorescence immunostaining of the tumor and nontumor tissue was performed with a sequential fluorescent method as described17 (link). Immunofluorescence was observed with the Olympus IX-71.