Immunohistochemistry

For immunohistochemical analysis, formalin-fixed paraffin-embedded tissue sections were de-paraffinized with xylene and rehydrated with ethanol solutions and distilled water. Antigen retrieval was performed by heating the sections in EDTA antigen retrieval buffer (pH 9.0) at 99°C for 25 min. Then the slides were washed in PBS (pH 7.4) three times for 5 min. After blocking in endogenous peroxidase with 3% hydrogen peroxide solution at room temperature for 25 min, the slides were again washed in PBS (pH 7.4) three times for 5 min. To block unspecific antibody binding, sections were incubated with 3% BSA (Solarbio, Beijing, China) for 30 min. Sections were then incubated overnight at 4°C in PBS (pH 7.4) containing primary antibodies, blood group Lewis a and b antibodies (Catalog number: sc-51512 and sc-51513, Santa Cruz Biotech, Texas, U.S.A.) at 1:20 in PBS (pH 7.4). Sections were washed three times in PBS then incubated with polyclonal goat anti-mouse immunoglobulins/HRP (Dako, Glostrup, Denmark) at room temperature for 50 min. The the slides were rinsed in PBS three times for 5 min and HRP was added. Rabbit/Mouse (DAB+). Harris staining was performed to highlight the nucleus of cells.

Lewis antigen expression was evaluated quantitatively using the Image Pro Plus 6.0 analysis system (Media Cybernetics, Silver Spring, MD) by calculating the mean density, which was integrated optical density (IOD) divided by area of interest [29]. Three 200X fields under microscope of each slide were randomly selected and the mean density was obtained for further statistic analysis.