WT controls and Fmr1 KO mice were anesthetized, perfused with 20 ml PBS and 20 ml 4% paraformaldehyde; and tissues were collected. Ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and cut to 20 μm sections. Slides were deparaffinized in xylene, rehydrated and H&E stain was performed to count ovarian follicles. For ovarian vasculature and innervation studies, frozen floating sections were stained with antibody to CD31 (1:2000 dilution, 553370, BD Biosciences) or with antibody to tyrosine hydroxylase (TH, 1:5000, ab112, Abcam) for 48 hours at 4°C, followed by overnight incubation with goat anti-rat IgG-Alexa 488 (1:2000, A11006, Vector Laboratories, Burlingame, CA) or goat anti-rabbit IgG-Alexa 488 (1:1000, A11034, Vector Laboratories, Burlingame, CA), respectively. Vascularization of corpora lutea and antral follicles was quantified by the mean fluorescent intensity (MFI) using Fiji ImageJ. Ovarian innervation was quantified by counting the number of neuronal projections in direct contact with follicles or corpora lutea.
Hypothalami were sectioned to 100 μm sections. Sections containing organum vasculosum laminae terminalis (OVLT) where GnRH neurons are located, were blocked and stained for GnRH using rabbit anti-GnRH antibodies (1:10,000 dilution) kindly provided by Greg Anderson (University of Otago; Dunedin, New Zealand (62 (link))), GABAγ2 receptor subunit (1:10,000 dilution, guinea pig anti-GABAγ2, Synaptic systems 224 004), VGAT (1:5,000, mouse anti-VGAT, Synaptic systems 131 011) for 72 hours at 4°C. After PBST washes, slides were incubated overnight at 4°C with secondary antibodies goat anti-rabbit IgG-Alexa 488 (1:1000, A11034, Vector Laboratories, Burlingame, CA); anti-mouse IgG-Alexa 594 (1:1000, A11032, Vector Laboratories, Burlingame, CA); anti-guinea pig–biotin (1:1000, BA-7000) followed by streptavidin-Cy5 (1:1000, 434316, Vector Laboratories, Burlingame, CA). Secondary antibody-only controls were performed to determine antibody specificity. To determine puncta density, we followed our established protocol as previously published (60 (link), 63 (link)–66 (link)). Puncta were counted in the individual neurons, by an investigator blinded to the group, where at least 45 μm of the axon proximal to soma can be observed using z-stack acquired by confocal Leica SP2 microscope. At least 15-20 individual neurons from 4-5 different sets of mice were counted. 3–D reconstruction was performed by Imaris software (Bitplane, Inc; Concord, MA).
Immunostaining for FMRP was performed using antigen retrieval methods, as previously described (67 (link)). Slices were stained overnight with mouse anti-FMRP (1:1000; Developmental Studies Hybridoma Bank, catalog #2F5-1-s, RRID: AB_10805421). Secondary antibody was donkey anti-mouse Alexa 594 (1:300; Molecular Probes, A-21202). Slices were mounted on slides with Vectashield mounting medium containing DAPI (Vector Laboratories, H-1200).
Hypothalami were sectioned to 100 μm sections. Sections containing organum vasculosum laminae terminalis (OVLT) where GnRH neurons are located, were blocked and stained for GnRH using rabbit anti-GnRH antibodies (1:10,000 dilution) kindly provided by Greg Anderson (University of Otago; Dunedin, New Zealand (62 (link))), GABAγ2 receptor subunit (1:10,000 dilution, guinea pig anti-GABAγ2, Synaptic systems 224 004), VGAT (1:5,000, mouse anti-VGAT, Synaptic systems 131 011) for 72 hours at 4°C. After PBST washes, slides were incubated overnight at 4°C with secondary antibodies goat anti-rabbit IgG-Alexa 488 (1:1000, A11034, Vector Laboratories, Burlingame, CA); anti-mouse IgG-Alexa 594 (1:1000, A11032, Vector Laboratories, Burlingame, CA); anti-guinea pig–biotin (1:1000, BA-7000) followed by streptavidin-Cy5 (1:1000, 434316, Vector Laboratories, Burlingame, CA). Secondary antibody-only controls were performed to determine antibody specificity. To determine puncta density, we followed our established protocol as previously published (60 (link), 63 (link)–66 (link)). Puncta were counted in the individual neurons, by an investigator blinded to the group, where at least 45 μm of the axon proximal to soma can be observed using z-stack acquired by confocal Leica SP2 microscope. At least 15-20 individual neurons from 4-5 different sets of mice were counted. 3–D reconstruction was performed by Imaris software (Bitplane, Inc; Concord, MA).
Immunostaining for FMRP was performed using antigen retrieval methods, as previously described (67 (link)). Slices were stained overnight with mouse anti-FMRP (1:1000; Developmental Studies Hybridoma Bank, catalog #2F5-1-s, RRID: AB_10805421). Secondary antibody was donkey anti-mouse Alexa 594 (1:300; Molecular Probes, A-21202). Slices were mounted on slides with Vectashield mounting medium containing DAPI (Vector Laboratories, H-1200).
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