Histofine simple stain max po r kit

Biopsy tissue was fixed in formalin and embedded in paraffin, and then 4-µm thin-sections were prepared. Four cases with p53 wild-type (1T, M11, 7T, M5) and 4 cases with a p53 mutation (M9, M8, M13, 8T) were used. After deparaffinization and removal of endogenous peroxidase, antigen activation was performed using citrate buffer (pH 6) in a 600 W microwave oven for 5 min [cystain A (CSTA), stratifin (SFN) or an autoclave for 5 min desmocollin 3 (DSC3)].
The primary antibodies used were rabbit polyclonal anti-human CSTA IgG (0.1 µg/ml, HPA001031; Atlas Antibodies AB, Stockholm, Sweden), mouse monoclonal anti-human 14-3-3 sigma IgG1 clone 1.N.6 (1 µg/ml, GTX14123; GeneTex, Irvine, CA, USA), mouse monoclonal anti-human desmocollin 3 IgG1 clone Dsc3-U114 (0.05 µg/ml, 61093; Progen Biotech, Heidelberg, Germany), and mouse monoclonal anti-human p53 IgG2b clone DO-7 (0.69 µg/ml, M7001; Dako, Glostrup, Denmark). The reactions were carried out overnight at 4°C. A Histofine Simple Stain MAX-PO (R) kit or MAX-PO (M) kit (Nichirei, Tokyo, Japan) was used for secondary antibodies. The sections were colored with diaminobenzidine and nuclei were stained with hematoxylin.

The expression of phosphor‐MET was detected in deparaffinized formalin‐fixed, paraffin‐embedded tissue sections (4 μm thick) using an MET Ab. Proliferating cells were detected by incubating tissue sections with a Ki‐67 Ab (Clone MIB‐1; DAKO Corp.). Apoptotic cells were detected by TUNEL staining of tissue sections using an in situ cell death detection kit (Roche Diagnostics GmbH). Antigens were retrieved by microwaving the tissue sections in 10 mmol/L citrate buffer (pH 6.0) and Tris‐EDTA buffer (pH 9.0). After incubation with a secondary Ab, peroxidase activity was visualized via the DAB reaction using Histofine Simple Stain MAX‐PO(R) kit (Nichirei). The sections were counterstained with hematoxylin. All sections were also stained with H&E.