Immunohistochemistry of MUC2, MUC5AC, MUC6, CDX2, Ki67, p53 and β-catenin was performed as previously described4 (link), 8 (link). The following antibodies were used: p53 (DO-7 + BP53-12; 1:2,000; Thermo scientific, PA, USA), β-catenin (Clone 14; 1:5,000 BD Transduction Laboratories, CA, USA), MUC2 (Ccp58; 1:500; Novocastra, Newcastle, UK), MUC5AC (CLH2; 1:100; Novocastra, Newcastle, UK), MUC6 (CLH5; 1:50; Novocastra, Newcastle, UK), CDX-2 (Clone EPR2764Y, Immunologic, Duiven, The Netherlands) Ki 67 (MIB-1; 1:100; Immunotech Marseille, France)
In brief, 4μm sections were deparaffinized in xylene and endogenous peroxidase was blocked in 0.3% H2O2 (Merck, NJ, USA) in methanol. Antigen retrieval was performed by cooking for 20 minutes in 10/mM Tris/EDTA buffer. A blocking step was performed with 5% normal goat serum in PBS for 10 minutes. Next, the primary antibody was incubated for 1 hour at room temperature (p53) or overnight at 4°C (MUC2, MUC5AC, MUC6, CDX2, Ki67, and β-catenin). Staining of the antibodies was done with the Poly-HRP-Goat α Mouse/Rabbit IgG (Immunologic, Duiven, The Netherlands) and 3,3-diamino-benzidine (DAB, Sigma Aldrich, MO, USA). Sections were lightly counterstained with hematoxylin.
In brief, 4μm sections were deparaffinized in xylene and endogenous peroxidase was blocked in 0.3% H2O2 (Merck, NJ, USA) in methanol. Antigen retrieval was performed by cooking for 20 minutes in 10/mM Tris/EDTA buffer. A blocking step was performed with 5% normal goat serum in PBS for 10 minutes. Next, the primary antibody was incubated for 1 hour at room temperature (p53) or overnight at 4°C (MUC2, MUC5AC, MUC6, CDX2, Ki67, and β-catenin). Staining of the antibodies was done with the Poly-HRP-Goat α Mouse/Rabbit IgG (Immunologic, Duiven, The Netherlands) and 3,3-diamino-benzidine (DAB, Sigma Aldrich, MO, USA). Sections were lightly counterstained with hematoxylin.