Histofine simple stain max po m kit

For the assessment of p53 expression, 4-μm-thick sections were cut and mounted on a silane-coated glass slide, deparaffinized, and soaked for 15 min at room temperature in 0.3% H 2 O 2 /methanol to block endogenous peroxidases. Immunostaining was performed with mouse anti-p53 monoclonal antibody (DO7; 1:50; Catalog No.M7001, Dako) at 4 °C overnight. The primary antibody was visualized using the Histofine Simple Stain MAX-PO(M) Kit (Nichirei, Tokyo, Japan) according to the manufacturer's instructions. The slide was then counterstained with hematoxylin. Lesions, in which all or most cells showed dark brownnuclear staining, were considered positive [26] (link). All samples were observed at a magnification of 400 × . Samples with 10% or higher staining for p53 in the nucleus were considered p53-positive.

The polymer peroxidase method was used for immunohistochemical staining of oesophageal biopsies. As the primary antibody, human basophil-specific antibody (BB1, antibasogranulin Ab, mouse IgG2a) at 1:10 dilution was used to detect basophils 16 and antitryptase (mouse IgG2a; Santa Cruz Biotechnology, Inc., TX, USA) at 1:500 dilution were used to detect mast cells. Formalin-fixed, paraffin-embedded sections (5 lm) were deparaffined with xylene, dehydrated with ethanol, pre-treated with 0.1% trypsin in 50 mM Tris-HCl containing 0.1% CaCl 2 for 37 °C at 30 min, and followed by additional treatment with 0.1% saponin in a phosphate-buffered saline (PBS) buffer for 30 min at room temperature. After immersion of the sections in a blocking agent solution (protein block serum free; Dako Japan, Tokyo, Japan) for 10 min, endogenous peroxidase was inactivated by immersion of sections in 3% hydrogen peroxide for 15 min. They were then incubated with BB1 antibody and a peroxidase-labelled anti-mouse polymer secondary antibody (Histofine Simple Stain MAX-PO (M) kit; Nichirei Biosciences Inc., Tokyo, Japan) for 30 min, stained with a diaminobenzidine chromogen (Nichirei), and counterstained with haematoxylin. 17 Stained slides were dehydrated and coverslipped. Mast cells were stained using the same protocol as described above, but proteinase K (Dako) was used as an antigen retrieval buffer.