Histofine simple stain po m kit

4-μm sections cut from the paraffin blocks were mounted on silane-coated glass slides. This was followed by deparaffinization and soaking for around 25 min in H₂O₂/methanol (0.3%) at room temperature. The sections were subsequently heated in hot water and Immunosaver (Nishin EM, Co., Ltd., Tokyo, Japan) at 98°C for 50 min. Non-specific binding sites were blocked by incubating with Protein Block serum-free for 30 min. A mouse monoclonal anti-LNC1 antibody was applied at a dilution of 1 : 100 for 24 h at 4°C. The primary antibody was visualized using the Histofine Simple Stain PO (M) kit (Nichirei, Tokyo, Japan), as per the manufacturer’s guidelines. Chromogen 3,3′-diaminobenzidine tetrahydrochloride containing 0.005% H₂O₂ in 50 mM ammonium acetate-citrate acid buffer (pH 6.0) was applied as 0.02% solution. The sections were lightly counterstained with Mayer’s hematoxylin and mounted.