Anti estrogen

Dissected brain region homogenized in specialized lysis buffer, containing 25 mM HEPES, 0.1% Tween-20, 5 mM magnesium chloride, 1.3 mM EDTA, 1 mM EGTA and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, 1 μM pepstatin). Anti-estrogen, anti-aromatse and anti-TNFα mouse monoclonal antibodies [Abcam plc (Cambridge, UK)] used to detect the level of the respective molecules in brain lysate and in blood plasma. The Estrogen and TNF-α level in FC lysates were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA). The OD values were read in a microplate reader at 450 nm. The concentrations of TNF-α and estrogen in the sample were calculated against the standard curve generated using recombinant TNF-α.

Immunoprecipitation was done according to Kundu et al., 200952 . Briefly, about 100 μg of protein from the cortical list of of both male and female mice were immunoprecipitated using 10 μl each of anti-estrogen, anti-ERβ separately [Abcam plc (Cambridge, UK)] for overnight at 4 °C with gentle rotation. 25 μl Protein G CL-Agarose (Bangalore Genei, India) was added to the previous mixture, depending on the experiment and allowed it to mix for 4 hours at 4 °C with gentle rotation. It was then centrifuged at 3000 RPM for 2 min. The immunoprecipitates were washed extensively with sterile PBS and separated by SDS-PAGE, followed by western analyses with anti-ERα, anti-ERβ, RelB, p65, antibody in separate cases as described above.