Anti endothelin 1 antibody

Cells were harvested and cell lysate were prepared by incubating the cells in RIPA (radio-immunoprecipitation assay) buffer containing protease inhibitor cocktail (10 µl/ml) (Roche Molecular Biochemicals, Mannheim, Germany) for 30 minutes on ice. Total protein concentrations were determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Cell lysate (20 µg total protein per sample) were subjected to SDS-PAGE followed by transferring to PVDF membrane. The membrane was incubated for 1 hour in blocking buffer, probed with the primary antibody for 1 hour at room temperature, and then washed twice with blocking buffer without milk. The membrane was then incubated with the appropriate secondary antibody for 1 hour at room temperature. After washing with blocking buffer, the protein bands were visualized using the Western Lightning Plus-ECL Reagent (Perkin Elmer, Waltham, MA, USA). Images of the blots were captured using the BioSpectrumAC Imaging System (UVP, Upland, CA, USA). The band intensity was quantified using UVP Visionworks LS software.
The anti-Endothelin 1 antibody was purchased from Abcam (Cambridge, MA, USA) and the anti-β actin antibody from Sigma-Aldrich (St. Louis, MO, USA). The mouse HRP-conjugated secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

To confirm ET-1 and ET_AR expressions in tissue samples from OSCC patients, we performed immunohistochemical staining. Each tissue sample was fixed in 10% buffered formalin (Sigma-Aldrich Japan, Tokyo), embedded in paraffin, and cut into serial sections (approx. 4 μm). After deparaffinization and antigen activation, immunostaining was performed using the catalyzed signal amplification (CSA) method. As the primary antibodies, Anti-Endothelin 1 antibody (Abcam, Cambridge, MA) and Anti-Endothelin A Receptor antibody (Abcam) diluted 500-fold in phosphate-buffered saline (PBS) were used. To evaluate the specificity of the staining, we used Universal Negative Control for IS-Series Rabbit Primary Antibodies (Dako Japan, Tokyo) as a negative control. For chromogenic detection, 3, 3′-diaminobenzidene tetrahydrochloride (DAB) was used. As a counterstain, Mayer’s hematoxylin was used.