Goat anti agrp

Brain tissue sections were washed 3X in PBS prior to a blocking step containing 3% normal donkey serum and 0.4% Triton X-100 in PBS for one hour at room temperature. Primary antibody was prepared in the same blocking solution and incubated overnight at the following concentrations: Rabbit anti-HSD2 (H-145; Santa Cruz Biotechnology) 1:300, Rabbit anti-TH (EMD Millipore) 1:3,000, Rat anti-mCherry (Life Technologies) 1:3,000, Chicken anti-GFP (Life Technologies) 1:3,000, Rabbit anti- c-Fos (EMD Millipore) 1:3,000, Rabbit anti-CGRP (Peninsula Labs) 1:1,000, Goat anti-AgRP (Neuromics) 1:3,000, and Sheep anti-FoxP2 (R&D Systems) 1:1,000. The next day sections were washed 5X in PBS, then incubated for 2 hours at room temperature in Alexa Fluor fluorescent secondary antibody (Life Technologies; 1:1,000) prepared in blocking solution. Finally, sections were washed 3X in PBS, mounted on gelatin-coated slides, and coverslipped with Vectashield mounting media containing DAPI (Vector Labs). Fluorescent images were captured using an Olympus VS120 slide-scanning microscope. In NTS^HSD2 neuron ablation experiments (Figure 5B,C), one of three serial sections was used for posthoc histological analysis of HSD2 and TH immunoreactive neurons.

Anesthesized mice were perfused with 4% (w/v) PFA. Brains were postfixed in 4% PFA, and immersed in 30% sucrose overnight and sectioned at 10 μm on a cryostat, except Figures 3A, 3C & 7, where 35-μm sections were used. Immunohistochemistry was performed using the following antibodies: goat anti AgRP (1:1000, Neuromics), rabbit anti DCX (1:500, Abcam), rat anti BrdU (1:200, AbD serotec), mouse anti NeuN (1:200, Chemicon), mouse anti HuC/D (1:2000, Invitrogen) and mouse anti nestin (1:200, Millipore). For staining of DCX, HuC/D, and NeuN, sections were subjected to heat-mediated antigen retrieval in a 10 mM citrate solution. For BrdU staining, sections were incubated in a 1N hydrochloric acid solution at 55°C for 3 minutes. Sections were incubated in a blocking solution followed by primary antibody overnight at 4°C and washed. A solution of secondary antibodies was then applied for 1 hour at room temperature. Images were acquired with an Olympus BX51WI microscope equipped with a QImaging Retiga 2000R digital camera. Double-immunofluorescence analyses of BrdU and NeuN, HuC/D or nestin were done by applying the two different antibodies sequentially. Pictures taken before and after immunohistochemistry were merged in Adobe Photoshop software using anatomical landmarks.

Histology was performed as previously described^{44 (link)}. We used the following primary antibodies: rabbit anti-dsRed, Clonetech (632496) 1:1,000; chicken anti-GFP, Life Technologies (A10262) 1:1,000; goat anti-AgRP, Neuromics (GT15023) 1:1,000; rabbit anti-cfos, Santa- Cruz (sc-52) 1:1,000.