Immediately after excision, the specimens were fixed in 10% neutral buffered formalin, embedded in paraffin wax, cut into 3-μm-thick paraffin sections, and stained with hematoxylin and eosin for routine pathological diagnosis. For immunohistochemical staining, additional 3-μm-thick sections were cut from paraffin-embedded tissues and placed on poly-l -lysine-coated glass slides. To evaluate the expression of mucins, annexin A10, and mismatch repair proteins, immunostaining was performed using antibodies against MUC2 (Ccp58, Novocastra Laboratories, Newcastle, UK), MUC6 (CLH5, Novocastra Laboratories), MUC5AC (CLH2, Novocastra Laboratories), annexin A10 (NBP1-90156, Novus, Litteleton, CO, USA), CD10 (56C6, Novocastra Laboratories), hMLH1 (clone G168-15; BD Biosciences, Bedford, MA, USA), and hPMS2 (clone C-20; Santa Cruz Biotechnology, Dallas, TX, USA).
Immunohistochemistry was performed using the DAKO Envision + system, consisting of dextran polymers conjugated with horseradish peroxidase (DAKO), USA. The specimens in citrate buffer (pH 6.0) were heated by microwave at 750 W three times for 5 min each before incubating with the antibodies (H2500, Microwave Processor, Bio-Rad, USA), as described previously. Hematoxylin was used as the counterstain.
Immunohistochemistry was performed using the DAKO Envision + system, consisting of dextran polymers conjugated with horseradish peroxidase (DAKO), USA. The specimens in citrate buffer (pH 6.0) were heated by microwave at 750 W three times for 5 min each before incubating with the antibodies (H2500, Microwave Processor, Bio-Rad, USA), as described previously. Hematoxylin was used as the counterstain.