Immunohistochemical Analysis of Ovarian Receptors

Sections of each ovary sample (slides: three for every group) were made for Immunohistochemical (IHC) examinations of FSHR and LHR using a previously described method^{41 (link)}. Briefly, the sections were covered with 0.5% TritonX-100 in phosphate buffer saline (PBS) for 20 min. Endogenous peroxidase signals were blocked with 3% hydrogen peroxide in methanol for 5 min at room temperature. 5% bovine serum albumin (BSA: Sigma-Aldrich, Cat: A9647) diluted in 0.1 M PBS (pH 7.2) was utilized to block the non-specific background staining for 1 h, and incubated with anti-FSHR and anti-LHR antibodies overnight at 4 °C. The primary antibody for each protein was diluted in PBS (PH = 7.4) at the following dilutions: FSHR (Biosynthesis Biotechnology, Beijing, China) 1:400; LHR (Biosynthesis Biotechnology, Beijing, China) 1:400. In negative control sections, PBS substituted the primary antibody-containing solution. On the following day, the sections were cleaned 3 times in PBS for 5 min, incubated with biotinylated anti-rabbit IgG antibody (for FSHR and LHR, Birmingham, USA) diluted 1:200 in PAV buffer (containing 0.1 M PBS, 0.1% BSA, and 0.05% thimerosal) for 50 min at 4 °C. Thereafter, the sections were washed 3 times in PBS for 5 min, and visualized using diaminobenzidine tetrahydrochloride (DAB). Next, they were rinsed and counterstained with Mayer's hematoxylin. Then, the sections were dehydrated and covered with a mounting medium (DPX; POCh, Gliwice, Poland). Finally, the sections were examined by two independent observers using an optical microscope. Semi-quant was assessed to estimate the expression level of LHR and FSHR in the ovary for every group taking advantage of the software Image-pro plus 6.0 (Media Cybernetics, Washington, USA)^{42 (link)}. Approximately 3 fields were examined per slide, and 3 slides were analyzed per group. Ovarian tissue exposed to secondary antibodies only was used as a negative control.