Envison hrp

Tissue specimens were surgically obtained and fixed in 10% formalin for 24 h at room temperature, followed by paraffin embedment at 56°C. From each tissue block, 4-μm thick sections were cut, mounted on MAS-coated slide glasses (Matsunami Glass Ind., Tokyo, Japan), and de-paraffinized in xylen. The sections were rehydrated in graded ethanol solutions. For antigen retrieval of tissue sections, the slides were incubated with Proteinase K (DAKO) for 5 min at room temperature in a moist chamber. Endogenous peroxidase was blocked with 3% hydrogen peroxide (H 2 O 2 ) for 10 min at room temperature and the slides were washed thrice with PBS. The sections were blocked with 5% BSA in PBS and incubated with primary antibody (rabbit polyclonal anti-human LF; Abcam, UK) at 4°C overnight. The slides were washed thrice with PBS and probed with a secondary antibody (EnVison/HRP; DAKO, Japan) according to the instructions supplied by the manufacturers. Visualization was performed with 3′, 3′-diaminobenzidine substrate solution. The slides were counterstained with hematoxylin 3G (Sakura-finetek, Tokyo) for 45 s at room temperature. LF immunoreactivity was classified into 2 categories: positive, ≥ 10% of tumor cells were immunostained; negative, < 10% of tumor cells were immunostained.