Diluent buffer

Soluble HLA-G concentrations were evaluated by a specific sandwich ELISA in plasma using MEM-G/9
[23 (link)] and anti-human β2-microglobulin as capture and detection antibodies respectively
[24 (link)]. Microtitration plates (Corning Incorporated, USA) were coated overnight at 4°C with 10 μg/mL MEM-G/9 Mouse-anti-human HLA-G mAb (ExbioPraha, Czech Republic). Plates were saturated using 300 μl diluent buffer (DAKO, USA) ready to use for 2 h. All samples were previously centrifuged and pre-diluted (½) in diluent buffer. Plasma samples were tested in duplicate and incubated for 2 h. Plates were incubated for 1 h more with detection antibody (Rabbit-anti-human β2-microglobulin: (DAKO, USA)). Then the plates were incubated for 1 h with envision buffer (DAKO, USA) to obtain anti-β2-microglobulin horseradish peroxidase complex aimed to improve the efficiency of the reaction. All incubation steps were performed at room temperature and followed by four washes using washing buffer (H2O, PBS 1X, 0.1% Tween 20). The plates were incubated for 30 min with the substrate (Tetramethylbenzidine (TMB), Sigma Aldrich, USA) and absorbance was measured at 490 nm after adding HCL (1 N). Total sHLA-G levels were determined from a five-point standard curve (12.5-200 ng/ml) using dilutions of calibrated HLA-G5 purified from M8-HLA-G5 cell line culture supernatant and results were expressed as ng/mL.

Sectioned ovarian tissues were deparaffinized in a 60 °C dry oven and by xylene and ethanol. Deparaffinized tissues were subjected to antigen retrieval by EDTA (eLbio, Seongnam-si, Republic of Korea) reaction and slowly cooled with water. The ovarian tissues were washed with distilled water (D. W) and treated with peroxide blocking solution containing 3% H₂O₂ in methanol for 10 min. Next, the ovarian tissues were washed with D.W and treated with primary antibodies with diluent buffer (Dako) at 4 °C overnight. The rabbit anti-PDGF receptor β antibody (28E1; 3169S, Cell Signaling Technology) was diluted 1:250. After removal of the unbound primary antibody, the tissues in slides were incubated with Dako Real EnVision HRP Rabbit/Mouse secondary antibody (Dako, California, USA) at room temperature for 1 h. The slides were incubated with DAB and counterstained with hematoxylin (Dako). After reaction, slides were rinsed by tap water. Slides were dehydrated by ethanol and xylene. Tissues were analyzed by the 3D HISTECT program (The Digital Pathology Company).