Rabbit anti hsd2 h 145

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.