H2O2 for 10 min and 0.3% glycine, 0.3% Triton X-100 in K-PBS for 10 min. Brain sections were blocked in a K-PBS solution containing 1% Probumin (Millipore), 1% normal donkey serum (Jackson ImmunoResearch) and 0.03% Triton-X-100. Sections were incubated overnight at 4°C with primary antibody diluted in blocking solution. Tissue sections were washed and then incubated with Biotin-SP-conjugated Donkey Anti-Rabbit IgG (1:500, Jackson ImmunoResearch) in blocking solution for 1 hr. Sections were washed and incubated with Vectastain ABC (Vector Labs) for 1 hr. Staining was developed using a Nickel-enhanced diamino-benzidine reaction (Vector Labs). Brain sections were mounted on gelatin-subbed slides, dried, dehydrated and mounted with DPX (Electron Microscopy), unless the sections were also being processed for in situ hybridization. In this case, brain sections were mounted on SuperFrost Plus slides (Brain Research Laboratories) and processed for in situ hybridization, as described below. The primary antibodies used were P-STAT3 Y705 (1:1000, #9145, Cell Signaling Technologies), KISS1 (1:1000, #9754, Chemicon), CART (1:10000, #6838, PBL-Salk Institute), and TORC1 (1:2500, #6938, PBL-Salk Institute). For TORC1 IHC and IF the antibody was pre-adsorbed against the immunogen carrier β-thryoglobulin and 0.5 mM TORC2 peptide (MESPSTSL), which was synthesized at the Salk Institute Peptide Synthesis Core Facility.
