Sc33535

ETAR IHC performed as previously described. After deparaffinization, 4 µm paraffin sections of ovarium were incubated with 3% H₂O₂ in PBS for 5 minutes to reduce the endogenous peroxidase. Therefore, sections were incubated with anti-ETAR antibody (1:100) (SC33535, Santa Cruz) at 4°C for overnight and followed with EnVision+System+HRP anti-rabbit secondary antibody (K4002, Dako) (1:200) for 60 minutes at room temperature. Finally, immune-positive cells were visualized by incubating with 3,3’-diaminobenzidine. Hematoxylin staining used for counterstaining the sections.

Total cellular proteins were extracted from cell pellets with 20 mM Tris pH 8; 10% glycerol; 150 mM NaCl; 1% Triton X-100; 5 mM EDTA; 1 mM Na₃VO₄; 1 mM PMSF; 10 µg/mL aprotinin; 10 µg/mL leupeptin, then homogenized with four sonication cycles (20 seconds at 300 W). EDNRA and ENRB receptors were detected on nitrocellulose membrane (Biorad), pre-incubated in TBST-milk (Tris pH 7.6, NaCl, 0.05% Tween 20, 5% non-fat dry milk), by overnight incubation at 4 °C with rabbit anti- EDNRA (SantaCruz, sc-33535) or rabbit anti-EDNRB (SantaCruz, sc-33537) antibodies followed by incubation with anti-rabbit peroxidase conjugated IgG antibodies (Sigma, A0545). The β-actin was detected with mouse anti-β-actin-peroxidase antibody (Sigma, A3854, 1/50 000). Immunoreactive bands were revealed using “West Pico chemiluminescent substrate” (Thermoscientific).