Ab85543

Samples were fixed in 10% buffered formalin at room temperature for 1 day and embedded in paraffin. Paraffin sections (thickness, 4 µm) were deparaffinized, rehydrated in a graded alcohol series and washed in distilled water. Following antigen retrieval with citrate buffer (pH 6.0), endogenous peroxidase was quenched by incubating with hydrogen peroxide blocking reagent (cat. no. ab64218, Abcam) for 5–10 min at room temperature and sections were blocked with goat serum (cat. no. ab7481, Abcam) for 2 h at room temperature. Primary antibody against HPSE (1:200; cat. no. ab85543; Abcam) was then applied at 4°C overnight. Following washing, the sections were incubated with peroxidase-conjugated secondary antibodies against rabbit IgG (1:2,000; cat. no. ab205718, Abcam) for 1 h at room temperature. Color was developed with DAB solution (Dako; Agilent Technologies GmbH). The sections were counterstained with hematoxylin for 10 sec at room temperature prior to coverslipping. The sections were viewed under a light microscope (Olympus Corporation; magnification, ×40). For each section, ≥5 random fields were examined. The staining intensity of HPSE was scored as 0 (absent), 1 (weak), 2 (moderate) and 3 (strong) in a double-blinded manner. HPSE expression was categorized as low (score of 0–1) or high (score of 2–3).

The paraffin-embedded tissues were used for immunohistochemistry with heparanase (AB85543, Abcam, dilution 1:200), ET_AR (SC33535, Santa Cruz, dilution 1:100), and Ki-67 (CRM 325 B, Biocare Medical, dilution 1:100) antibodies. Paraffin sections, 4 μm in thickness, were placed on poly-L-lysine-coated slides. After deparaffinization, endogenous peroxidase was reduced by incubation with 3% H₂O₂ in phosphate buffer saline for 5 minutes. The secondary antibodies used were EnVision+System-HRP anti rabbit (K4002, Dako) for heparanase and the ET_AR and Histofine SAB-PO (MULTI) (414171F, Nichirei) for Ki-67. The chromogen used was 3,3’-diaminobenzidine. Hematoxylin was utilized for counterstaining.

Control and stimulated SUM149 cells were lysed in RIPA buffer containing protease inhibitor cocktail as we previously described [58 (link)]. Total protein content was determined with BCA method and 50 μg protein /lane was loaded into 10% SDS-PAGE. After the semi-dry transfer step, nitrocellulose membrane was blocked with 5% skimmed milk/TBST solution, washed thrice with TBST, and probed with anti-human heparanase antibody (1:500; ab85543, Abcam, UK) overnight at 4C°. Following 1 h incubation at room temperature with HRP-conjugated secondary antibody, the membrane was subjected to a chemiluminescence reaction and signal quantification was performed with NIH ImageJ software. Subsequently, the membrane was stripped and incubated with anti-GAPDH (1:1000, Santa Cruz Biotechnology, Texas, USA) as housekeeping controls for normalization.