Supporting Methods

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Supporting Methods




Supporting Methods

Double-Label Immunofluorescence. Animals were anesthetized deeply with sodium pentobarbital (200 mg/kg) and perfused intracardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.3). For visualization of gonadotropin-inhibitory hormone (GnIH), sections were washed in PBS, incubated in 0.5% H₂O₂, and incubated in normal goat serum in 0.1% Triton X-100 (PBT) for 1 h. Sections were then incubated for 48 h at 4°C in antiserum generated against white-crowned sparrow GnIH (PAC 123a) diluted at 1:100,000 with 0.1% PBT. After incubation in anti-GnIH, brains were incubated for 1 h in biotinylated goat anti-rabbit (1:300; Vector Laboratories), followed by incubation in avidin-biotin-horseradish peroxidase complex (ABC Elite kit, Vector Laboratories). Brains were then incubated in a biotinylated tyramide solution (0.6%) for 30 min. This protocol allowed for the amplification of the highly diluted anti-GnIH antiserum required for double-labeling with two antibodies generated in the same species. Cells were then labeled by using Cy-2 conjugated to streptavidin (The Jackson Laboratory) as the fluorophore. After labeling for GnIH, sections were incubated for 48 h in a guinea pig anti-gonadotropin-releasing hormone (GnRH) antibody (antigenic determinants are amino acids 6-10; Advanced ChemTech) diluted at 1:10,000 with 0.1% PBT. GnRH cells were labeled with Cy-3 donkey anti-guinea pig (The Jackson Laboratory) as the secondary antibody/fluorophore. Sections were mounted onto gelatin-coated slides and coverslips were applied after dehydration in a graded series of alcohols.

Several control experiments were conducted to ensure that staining was specific. First, one or both antibodies were preadsorbed with the appropriate ligand. To determine the specificity of the GnIH antibody, it was preadsorbed with GnIH peptides from rat, Siberian hamster, or white-crowned sparrow. We recently discovered that the rat, hamster, and avian peptides share a common C terminus targeted by the avian antibody (K.I., T.U., K.U., L.K., and K.T., unpublished observations). Second, one or both primary antibodies were eliminated and all steps followed exactly the same. The same general procedures were used for double labeling of FOS (Santa Cruz Biotechnology; 1:200,000 dilution, then amplified), estrogen receptor-a (Upstate Biotechnology; 1:40,000 dilution, then amplified), androgen receptor (Upstate Biotechnology; 1:100,000 dilution, then amplified), and GnIH (1:10,000 dilution second antibody, not amplified).

For in situ hybridization, the fixed sections were first rehydrated with PBS and treated with proteinase K (10 mg/ml) for 30 min, followed by postfixation with 4% paraformaldehyde in PBS for 10 min. After treating the sections in 0.2 M HCl for 20 min, the slides were kept in 40% deionized formamide in 4´ SSC (1´ SSC = 150 mM NaCl and 15 mM sodium citrate, pH 7.0) for 30 min. Hybridization was carried out at 50°C for 15–17 h with 200 ng/ml digoxigenin-labeled RNA probe (antisense or sense) mixture dissolved in 50% Sigma hybridization solution 1 and 40% formamide. After washing six times with 50% formamide-2´ SSC at 50°C for 10 min each time, the sections were treated with 1.5% blocking reagent (Roche Diagnostics) in TBS and incubated with alkaline phosphatase-labeled sheep antidigoxigenin antibody (1:1,000 dilution in the blocking solution; Boehringer Mannheim) for 1 h. After this, the sections were washed three times with 0.05% Tween in TBS for 10 min each time. Immunoreactive products were detected by immersing the sections for 12 h in a substrate solution [0.035% nitro blue tetrazolium and 0.018% 5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris•HCl/100 mM NaCl (pH 9.5)], and the expression of GnIH mRNA was observed by using a Zeiss M1 microscope. Control for specificity of in situ hybridization of GnIH mRNA was performed by using a digoxigenin-labeled sense RNA probe complementary to the antisense probe sequence.