Following the detection of kisspeptin or NKB immunoreactivity, GnRH neurons were detected with a new guinea pig antiserum to GnRH (#1018; 1:50,000). The primary antibodies were reacted with biotin-SP–anti-guinea pig IgG (Jackson ImmunoResearch; 1:500; 1 h) and ABC (1:1000; 1 h) and then, immunoreactivity was visualized with DAB chromogen alone.
To generate the #1018 antiserum, an immunization antigen was prepared by conjugating 4 mg mammalian GnRH to 25 mg bovine thyroglobulin with 12 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in 4 ml 100 mM MES buffer (pH 4.7). Unconjugated peptides were removed from the antigen solution by dialysis against 0.1 M phosphate buffered saline (PBS, pH 7.6; 3 × 8 h). Two adult female guinea pigs were immunized intradermally (i.d.) with 10 μg antigen in 500 μl solution. For initial immunization, 10 μg antigen complex in 250 μl PBS was emulsified with an equal volume of Freund’s complete adjuvant (Sigma). Subsequent boosts with Freund’s incomplete adjuvant were administered at 2-week intervals. Antisera were sampled from the ear 1 week after each booster injection and tested at various dilutions on tissue sections of the mouse preoptic area (for GnRH cell bodies) and median eminence (for GnRH axons). Upon achievement of the appropriate serum titer (3 months after the beginning of immunization), the animals were exsanguinated. The blood was allowed to clot and serum was collected. Sodium azide was added at 0.1% and antiserum aliquots were transferred to −20°C for long-term storage. Antibody samples were tested with immunohistochemistry on paraformaldehyde-fixed mouse and human tissue sections. For peroxidase-based detection of GnRH, various dilutions of the primary antibodies were made with 2% normal horse serum in PBS and applied to the sections for 16–48 h. The primary antibodies were reacted with biotinylated secondary antibodies (biotin-SP–anti-guinea pig IgG; Jackson ImmunoResearch Laboratories; 1:500) and the ABC Elite reagent for 60 min each. The peroxidase signal was visualized with Ni-DAB chromogen. As one positive control for the specificity of GnRH cell body labeling, the immunofluorescent detection of GnRH was performed on preoptic sections of GnRH-GFP transgenic mice in which GnRH neurons exhibit green fluorescence (Suter et al., 2000 (link)). As a second positive control also applicable to human test sections, dual-immunofluorescent visualization of GnRH immunoreactivity was carried out with one of the new guinea pig antisera (#1018 or #1035), in combination with a reference rabbit GnRH antiserum (LR1; 1: 5000) that was kindly provided by Dr. R. A. Benoit and used in previous publications from different laboratories, including our own (Hrabovszky et al., 2007 (link), 2010 (link)). In both control experiments, incubation with guinea pig GnRH antisera was followed by donkey anti-guinea pig-Cy3 (1: 1000; Jackson ImmunoResearch) for 5 h. The LR1 antibody was reacted with donkey anti-rabbit–FITC (1:200) for 5 h.
To generate the #1018 antiserum, an immunization antigen was prepared by conjugating 4 mg mammalian GnRH to 25 mg bovine thyroglobulin with 12 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in 4 ml 100 mM MES buffer (pH 4.7). Unconjugated peptides were removed from the antigen solution by dialysis against 0.1 M phosphate buffered saline (PBS, pH 7.6; 3 × 8 h). Two adult female guinea pigs were immunized intradermally (i.d.) with 10 μg antigen in 500 μl solution. For initial immunization, 10 μg antigen complex in 250 μl PBS was emulsified with an equal volume of Freund’s complete adjuvant (Sigma). Subsequent boosts with Freund’s incomplete adjuvant were administered at 2-week intervals. Antisera were sampled from the ear 1 week after each booster injection and tested at various dilutions on tissue sections of the mouse preoptic area (for GnRH cell bodies) and median eminence (for GnRH axons). Upon achievement of the appropriate serum titer (3 months after the beginning of immunization), the animals were exsanguinated. The blood was allowed to clot and serum was collected. Sodium azide was added at 0.1% and antiserum aliquots were transferred to −20°C for long-term storage. Antibody samples were tested with immunohistochemistry on paraformaldehyde-fixed mouse and human tissue sections. For peroxidase-based detection of GnRH, various dilutions of the primary antibodies were made with 2% normal horse serum in PBS and applied to the sections for 16–48 h. The primary antibodies were reacted with biotinylated secondary antibodies (biotin-SP–anti-guinea pig IgG; Jackson ImmunoResearch Laboratories; 1:500) and the ABC Elite reagent for 60 min each. The peroxidase signal was visualized with Ni-DAB chromogen. As one positive control for the specificity of GnRH cell body labeling, the immunofluorescent detection of GnRH was performed on preoptic sections of GnRH-GFP transgenic mice in which GnRH neurons exhibit green fluorescence (Suter et al., 2000 (link)). As a second positive control also applicable to human test sections, dual-immunofluorescent visualization of GnRH immunoreactivity was carried out with one of the new guinea pig antisera (#1018 or #1035), in combination with a reference rabbit GnRH antiserum (LR1; 1: 5000) that was kindly provided by Dr. R. A. Benoit and used in previous publications from different laboratories, including our own (Hrabovszky et al., 2007 (link), 2010 (link)). In both control experiments, incubation with guinea pig GnRH antisera was followed by donkey anti-guinea pig-Cy3 (1: 1000; Jackson ImmunoResearch) for 5 h. The LR1 antibody was reacted with donkey anti-rabbit–FITC (1:200) for 5 h.
