Publication protocol
Sections were washed three times in PB 0.1M for 5 min each and then microwaved in 10 mM citrate buffer pH 6.0 (4 min at 800 W followed by 2 × 5 min at 400 W) for antigen unmasking. Sections were then washed in PB 0.1M, and incubated in blocking solution (5% donkey serum + 0.3% Triton X-100) in PB 0.1M for 60 min. Then, sections were incubated in rabbit anti-ERα (1/500; Millipore, Cat# 06–935 Lot#2708536, RRID:AB_310305) or guinea pig anti-GnRH (1/3000 raised by Dr. Erik Hrabovszky, Laboratory of Endocrine Neurobiology, Institute of Experimental Medicine of the Hungarian Academy of Sciences, Budapest, Hungary) and sheep anti-nNOS (1/10,000; generous gift from Dr. P. C. Emson, Medical Research Council, Laboratory for Molecular Biology, Cambridge, UK) and, when required, chicken anti-GFP antibody (1/500; Abcam, Cat# ab13970 Lot#GR236651–14 RRID:AB_300798) in blocking solution for 48 hr at 4°C. After incubation in the primary antibody, sections were rinsed with PB 0.1M three times for 10 min each and then incubated in the secondary antibody: Alexa-568-conjugated donkey anti-rabbit or anti-guinea pig (Thermo Fisher Scientific Cat# A10042, RRID:AB_2534017), Alexa-647-conjugated donkey anti-sheep (Thermo Fisher Scientific Cat# A-21448, RRID:AB_2535865) and donkey anti-chicken Alexa 488 (Jackson ImmunoResearch Labs Cat# 703–545-155, RRID:AB_2340375) for 60 min at room temperature. Sections were then washed, counterstained with Hoechst (1/10,000; Thermo Fisher Scientific Cat# H3569, RRID: AB_2651133) for 5 min, washed twice for 5 min and coverslipped with Mowiol coverslip mounting solution.
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