Biotinylated donkey anti rabbit igg

For the double-label study, brain sections were processed for nNOS/RFP double-fluorescence labeling. After two hours of incubation in the blocking buffer, the sections were incubated overnight in a combination of mouse anti-nNOS (1:1,000; Sigma-Aldrich) and rabbit anti-RFP (1:1,500; Rockland Immunochemicals, Gilbertsville, PA) antibody. The sections were then rinsed in PBS, incubated in a mixture of donkey DyLight 488 anti-mouse IgG (1:500; Jackson ImmunoResearch, West Grove, PA) and biotinylated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch, West Grove, PA), again rinsed in PBS, and incubated in DyLight 594 streptavidin conjugate (1:500; Jackson ImmunoResearch, West Grove, PA). After rinsing in PBS, all sections were mounted on gelatin-coated slides and coverslipped using Fluoromount mounting media (Electron Microscopy Sciences, Hatfield, PA).

AR immunoreactivity was visualized using a modified tyramide signal amplification (TSA) method previously described ^{42 (link)}. Brain sections were rinsed with 0.1M PBS, incubated in 0.6% hydrogen peroxide for 30 min, rinsed with 0.1M PBS, then blocked with 3% normal donkey serum with 0.25% Triton-X-100 for 1 h at room temperature. Sections were incubated overnight with rabbit anti-AR antibody (1:200, AbCam [EPR1535(2)], Cat #ab133273, RRID: AB_11156085). A series with no primary antibody was included as a negative control (Figure 1A-B). Sections were rinsed with 0.1M PBS and then incubated for 1 h with biotinylated donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch Laboratories, Cat #711–065-152, RRID: AB_2340593), followed by incubation in avidin-biotin (AB) solution in 0.1M PBS (1:1000, Vector Laboratories) for 1 h. Next, sections were incubated in biotinylated tyramide (1:250, Perkin Elmer) with 0.009% hydrogen peroxide for 10 min, followed by incubation with streptavidin-conjugated AlexaFluor 594 (1:1000, Invitrogen, ThermoFisher) for 1 h. Sections were mounted onto gelatin-coated slides and coverslipped with ProLong Gold Antifade mounting medium (Invitrogen, ThermoFisher).

To identify the brain regions where refeeding-activated neurons are contacted by PB neurons, triple-labeling immunofluorescence for PHA-L, c-Fos and the neuronal marker, HuC/HuD, was performed on sections of all animals (n=5) with a PHA-L injection site confined within the area of the PB. Following pre-treatment as described above, the sections were incubated in anti-PHA-L serum at 1:5,000 for 2 days at room temperature, followed by biotinylated donkey anti-rabbit IgG (Jackson Immunoresearch) diluted to 1:500 and then ABC (1:1,000) for 1 h after rinses in PBS. The immunoreaction was amplified with biotinylated tyramide as above, and after further washes, the sections were incubated in streptavidin-conjugated Alexa 555 (Vector) at a 1:500 dilution. Then, the sections were incubated in a mixture of rabbit antiserum against c-Fos at 1:2,000 dilution and mouse antiserum against HuC/HuD (Molecular Probes) at 1:500 for 2 days at 4°C. After washes in PBS, the sections were immersed in a cocktail of fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (Jackson) at 1:250 dilution and Cy5-conjugated donkey anti-mouse IgG (Jackson) at 1:100 dilution and incubated for 2 h at room temperature. The sections were then mounted onto glass slides and coverslipped with Vectashield Mounting Medium.