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CK immunohistochemical staining was performed using cocktails of antibodies against CK13 and CK17, and against CK13 and CK17+14. All specimens were fixed in 10% formalin, embedded in paraffin, and cut into 3-��m slices from the paraffin blocks. Sections were deparaffinized with xylene and rehydrated ethanol, and treated with 0.3% hydrogen peroxidase in methanol for 30?min to block endogenous peroxidase activity. Antigen retrieval was performed Temsirolimus (CCI-779, NSC 683864) by boiling in 0.01?mol/L of citrate buffer at pH?6.0 for 30?min. After three 5-min washes with phosphate-buffered saline (PBS), sections were treated with 5% normal goat serum for 30?min at room temperature to block nonspecific binding sites. All sections were incubated overnight at 4��C with cocktails of antibodies against CK13 and CK17, or against CK13 and CK17+14. Sections were incubated with Simple Stain MAX-PO (mouse) (Nichirei Bioscience, Tokyo, Japan) for 30?min. After three 5-min washes with PBS, sections were incubated with Simple Stain AP (rabbit) (Nichirei Bioscience) for 60?min and rinsed three times with PBS for 5?min each. The peroxidase reaction was performed using 0.02% 3-3��-diaminobenzidine tetrahydrochloride for 10?min. After rinsing with distilled water and PBS, the alkali phosphatase reaction was performed using 4% new fuchsin (MP Biomedicals, Santa Selleckchem Pfizer Licensed Compound Library Ana, CA, USA) for 30?min. Positive staining for CK13 was brown, and positive staining for CK17 and CK17+14 was red. Finally, nuclear counterstaining was performed with Mayer's hematoxylin. Standard procedures were used for immunohistochemical staining with antibodies against Ki-67 and CK10.[18] Antigen retrieval was performed Apoptosis inhibitor for CK10 and Ki-67 antibodies by boiling in 0.01?mol/L of citrate buffer at pH?6.0 for 30?min. For Ki-67 antibody, antigen retrieval included additional digestion in 0.1% trypsin. The peroxidase reaction and nuclear counterstaining were performed as described above. Evaluation of the staining patterns was performed by the same three pathologists for all specimens. Staining for each CK was assessed as positive when more than 10% of the epithelial cells showed intracytoplasmic staining. Fisher's exact test was used to compare differences between the OKD and non-OKD specimens. P?