Anti muc6 clh5

Sections (4-μm-thick) were cut from formalin-fixed paraffin-embedded tissues, deparaffinized in xylene, and rehydrated in graded ethanol as reported previously [19] .
Endogenous peroxidase activity was blocked by incubating the samples with 3% hydrogen peroxide in methanol for 30 min. Antigen retrieval was achieved by microwaving the sections in citrate buffer for 20 min. Each section was incubated overnight at 4°C with the following primary antibodies: mouse monoclonal anti-MUC1 (Clone MA695, 1:100 dilution; Novocastra, Newcastle, UK), anti-MUC2 (CCP58, 1:100; Novocastra), anti-MUC5AC (CLH2, 1:150; Novocastra), anti-MUC6 (CLH5, 1:150; Novocastra), anti-CK 7 (OV-TL 12/30, 1:100; Dako, Carpinteria, CA, USA), anti-CK 20 (KS 20.8, 1:50; Dako), and anti-TS (TS106, 1:50; Dako) antibodies, and rabbit polyclonal anti-BNIP3 (clone ANA40, 1:200 dilution; Sigma-Aldrich, St. Louis, MO, USA) and anti-CDX2 (SC-19478, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies. The sections were sequentially incubated with a biotinylated anti-mouse or anti-rabbit immunoglobulin solution for 20 min followed by peroxidase-labeled streptavidin for 20 min. The resulting immunocomplexes were visualized with stable 3, 3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) as the chromogen. The sections were then rinsed with distilled water and counterstained with hematoxylin.