Rabbit anti erβ polyclonal antibody

Quercetin(4H-1-benzopyran-4-one,2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-f0lavone) and C₁₅H₁₀O₇·2H₂O (molecular weight 302.23, purity greater than 98%) were kind gifts from Swiss ALEXIS Biochemical Corporation. The structure of quercetin is shown in Figure 1, and its structure is similar with mammary estrogen 17b-estradiol (Figure 2), including a pair of hydroxy, the similar distance, and a phenol ring the latter one plays the decisive role in adsorbing on estrogen receptor. Gestrinone was purchased from Beijing ZiZhu Pharmaceutical (Beijing, China). Rat FSH and LH elisa Kits were purchased from USA Rapidbio Corporation. The mouse anti-ERa monoclonal antibody (68 kD), rabbit anti-ERβ polyclonal antibody (55 kD), and mouse anti-PR antibody [PR-AT 4.14]—ChIP Grade monoclonal antibody (99 kD) were all purchased from ABCAM company.

Immunohistochemistry procedure followed previously reported methods [23 (link)]. Briefly, 4 mm thick tissue section of vagina was mounted on polylysine-coated slides. The paraffin sections were dewaxed and incubated for 10 min with 3% hydrogen peroxide. Each section was incubated with blocking serum (Vectastain, ABC Kit) at room temperature for 30 min and then overnight at 4°C with rabbit anti-ERα polyclonal antibody (Abcam Biotechnology, UK) and rabbit anti-ERβ polyclonal antibody (Abcam Biotechnology, UK), respectively. Sections incubated in phosphate-buffered saline without antibody served as negative controls. Positive control experiments for ERα and ERβ were performed in adult Sprague-Dawley female rat's uterus. After incubation with biotinylated secondary antibody, sections were incubated with avidin-biotin complex reagent containing horseradish peroxidase for 30 min. The sections were then stained with 3,3'-diaminobenzidine (Sigma, USA). The Image-Pro Plus 6.0 System image analysis software was used for quantitative analysis.

Uterus was resuspended in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 m MEDTA, 0.1% sodium dodecyl sulfate, 0.5% NP-40) containing 10 mM phenylmethylsulfonyl fluoride and 2 mg/mL aprotinin. The protein was obtained to detect the levels of ERα and ERβ in target tissue by western blot. Western blot protocol and semiquantitative analysis were carried out as described in [24 (link)]. The rabbit anti-ERα polyclonal antibody (Abcam Biotechnology, UK) or rabbit anti-ERβ polyclonal antibody (Abcam Biotechnology, UK) was used. The experiment was done in triplicate. The relative quantity of each antibody was measured by Quantity One software. The density ratio of protein to GAPDH (Sangon Biotech, Shanghai, China) was calculated from the band density.