Localization of proteins was assessed by immunohistochemistry. Antigens were retrieved using 2100 Antigen Retriever (Aptum Biologics Ltd., Southampton, UK) in Tris–EDTA buffer (pH 9, MKI67) or citrate buffer (pH6). Samples were washed in TBS with 0.1% Tween20 (TBST) (#P1379, Sigma-Aldrich) and blocked for 1 h in 3% BSA in TBST at room temperature (RT). Antibodies against MKI67 (#MIB-1, Dako, diluted 1:500), GNRHR (#19950-1-AP, Proteintech, diluted 1:1000), LHCGR (clone 4G2, antibody donated by Dr Marco Bonomi); supernatant diluted 1:8 (Bonomi et al. 2006 (link)) or FSHR323 (donated by Dr N. Ghinea) (Vannier et al. 1996 (link)) at the concentration of 0.5 µg/mL, were applied on the slides and incubated overnight in 4°C. Endogenous peroxidase activity was quenched by 10-min incubation in 3% hydrogen peroxide (Sigma-Aldrich). Depending on the primary antibody host, Dako EnVision+ System-HRP polymer anti-mouse (K4007, Dako) or anti-rabbit (K4011, Dako) were applied, and visualized with Liquid DAB + Substrate Chromogen System (Dako). Slides were scanned by Pannoramic 250 Slide Scanner (3DHISTECH Ltd., Budapest, Hungary) and images were taken using Pannoramic Viewer (3DHISTECH Ltd.). The percentage of MKI67-stained cells was assessed using ImmunoRatio web application (http://153.1.200.58:8080/immunoratio/) (Tuominen et al. 2010 (link)) from four representative images of each sample.
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